C5a binding nucleic acids

ABSTRACT

The present invention is related to a nucleic acid molecule capable of binding to human C5a, wherein the nucleic acid molecule comprises a central stretch of nucleotides, wherein the central stretch of nucleotides comprises a nucleotide sequence of 5′ AUGn1GGUGKUn2n3RGGGHUGUKGGGn4Gn5CGACGCA 3′ [SEQ ID NO: 61], wherein n1 is U or dU, n2 is G or dG, n3 is A or dA, n4 is U or dU, n5 is U or dU and G, A, U, C, H, K, and R are ribonucleotides, and dU, dG and dA are 2′-deoxyribonucleotides.

The present invention is related to a nucleic acid molecule capable of abinding to C5a and/C5, the use thereof for the manufacture of amedicament, a diagnostic agent, and a detecting agent, respectively, acomposition comprising said nucleic acid molecule, a complex comprisingsaid nucleic acid molecule, a method for screening of an antagonist ofan activity mediated by C5a and/C5 using said nucleic acid molecule, anda method for the detection of said nucleic acid molecule.

The primary structure of the human anaphylatoxin C5a (complement factor5a; SwissProt entry P01031) was determined in 1978 (Fernandez & Hugli1978). It consists of 74 amino acids accounting for a molecular weightof 8,200 Da while the carbohydrate portion accounts for approximately3,000 Da. The carbohydrate portion of C5a exists as a single complexoligosaccharide unit attached to an asparagine at position 64. The threedisulfide bonds confer a stable, rigid structure to the molecule.

Although the three-dimensional structure of C5a forms from differentmammalian species has generally been maintained, the amino acid sequencehas not particularly well been conserved during evolution Sequencealignment demonstrates 64% overall sequence identity of human and mouseC5a. Human C5a shares the following percentages of identical amino acidswith C5a from:

Macaca mulatta (rhesus monkey) 85% Macaca fascicularis (cynomolgusmonkey) 85% Bos taurus (bovine) 69% Sus scrofa (pig) 68% Mus musculus(mouse) 64% Rattus norvegicus (rat) 61%

Besides the limited sequence homology, glycosylation is alsoheterogeneous. While human C5a is glycosylated on asparagine 64, themurine homologue is not glycosylated at all. The more distantly relatedhuman proteins C3a and C4a share only 35 and 40%, respectively, identitywith C5a.

The complement system was discovered at the beginning of the lastcentury as a heat sensitive serum fraction that “complemented” theantisera mediated lysis of cells and bacteria. Being a humoral componentof the natural unspecific (innate) immune response, it plays anessential role in host defence against infectious agents and in theinflammatory process. Complement can be activated via three distinctpathways (i) after an antibody attaches itself to a cell surface orbacteria (referred as classical pathway), (ii) directly by bacterial orviral glycolipids (referred as alternative pathway), or (iii) bycarbohydrates on bacteria (referred as lectin pathway). All theseactivation pathways converge at the point of activation of thecomplement components C3 and C5, where the common terminal pathwaystarts, culminating in assembly of the membrane attack complex (abbr.MAC). The complement system consists of more than 20 soluble proteinsthat function either as proteolytic enzymes or as binding proteins andmaking up about 10% of the total globulins in vertebrate serum. Inaddition, the complement system includes multiple distinct cell-surfacereceptors that exhibit specificity for proteolytic fragments ofcomplement proteins and that are expressed by inflammatory cells andcells regulating the adaptive immune response. There are severalregulatory proteins that inhibit complement activation and thus protecthost cells from accidental complement attack. The complement system canbecome activated independently or together with the adaptive immuneresponse.

The functions of complement include the process of opsonization (i.e.making bacteria more susceptible to phagocytosis), lysis of bacteria andforeign cells by inserting a pore into their membrane (referred asmembrane attack complex), generation of chemotactically activesubstances, increase of vascular permeability, evocation of smoothmuscle contraction, and promotion of mast cell degranulation. Similar tothe coagulation cascade, the process of complement activation isorganized in sequential enzymatic steps also known as an enzymaticcascade (Sim and Laich, 2000). The detailed sequence of theseinteractions is outlined in the following.

Classical Pathway. This antibody-dependent activation pathwaycomplements the specific antibody response. It is as elaboratelycontrolled as the alternative pathway, but lacks the spontaneousinitiation ability; i.e. the antibody-independent recognition function,and the feedback amplification mechanism. Among the activators of theclassical pathway are antigen-antibody complexes, β-amyloid, DNA,polyinosinic acid, polyanion-polycation complexes likeheparin/protamine, some enveloped viruses, monosodium urate crystals,lipid A of bacterial cell walls, plicatic acid, ant venompolysaccharide, subcellular membranes (such as mitochondria), as well ascell- and plasma-derived enzymes such as plasmin, kallikrein, activatedHageman factor, elastase or cathepsins. The antibody-induced classicalpathway starts with C1, which binds to the Fc-fragment of an antibody(IgM>IgG3>IgG1>>IgG2) ligated to a cell surface antigen. C1 is arecognition complex composed of 22 polypeptide chains in 3 subunits;C1q, C1r, C1s. C1q is the actual recognition portion, a glycoproteincontaining a collagen-like domain (exhibiting hydroxyproline andhydroxylysine residues) that looks like a bunch of tulips. Upon bindingvia C1q, C1r is activated to become a protease that cleaves C1s to aform that activates (by cleavage) both C2 and C4 to C2a/b and C4a/b. C2aand C4b combine to produce C4b2a, the C3 convertase (C3 activatingenzyme). C4a has only weak anaphylatoxin activity but is notchemotactic. C3 is central to all three activation pathways. In theclassical pathway, C4b2a convertase cleaves C3 into C3a/b. C3a is ananaphylatoxin. C3b combines with C4b2a to form C4b2a3b complex (C5convertase). C3b can also bind directly to cells making them susceptibleto phagocytosis (opsonization).

Alternative pathway. This pathway does not require antibodies foractivation and is of major importance in host defence against bacterialand viral infection because—unlike the classical pathway—it is directlyactivated by surface structures of invading microorganisms such asbacterial/viral glycolipids or endotoxins. Other activators are inulins,rabbit erythrocytes, desialylated human erythrocytes, cobra venomfactor, or phosphorothioate oligonucleotides. The six proteins C3,Factors B, D, H, I, and properdin together perform the functions ofinitiation, recognition and activation of the pathway which results inthe formation of activator-bound C3/C5 convertase. The cascade beginswith C3. A small amount of C3b is always found in circulation as aresult of spontaneous cleavage of C3 (“C3-tickover”), but theconcentrations are generally kept very low by subsequent degradation.However, when C3b binds to sugars on a cell surface, it can serve as anucleus for alternative pathway activation. Then Factor B binds to C3b.In the presence of Factor D, bound Factor B is cleaved to Ba and Bb; Bbcontains the active site for a C3 convertase. Next, properdin binds toC3bBb to stabilize the C3bBb convertase on the cell surface leading tocleavage of further C3 molecules. Finally, the alternative C5 convertaseC3bBb3b forms which cleaves C5 to C5a/b. Once present, C5b initiatesassembly of the membrane attack complex as described above. Generally,only Gram-negative cells can be directly lysed by antibody pluscomplement; Gram-positive cells are mostly resistant. However,phagocytosis is greatly enhanced by opsonization with C3b (phagocyteshave C3b receptors on their surface) and antibody is not alwaysrequired. In addition, complement can neutralize virus particles eitherby direct lysis or by preventing viral penetration of host cells.

Lectin pathway. The most recently discovered lectin or mannan-bindinglectin (abbr. MBL) pathway depends on innate recognition of foreignsubstances (i.e., bacterial surfaces). This pathway has structural andfunctional similarities to the classical pathway. Activation of thelectin pathway is initiated by the acute phase protein MBL, whichrecognizes mannose on bacteria, IgA and probably structures exposed bydamaged endothelium. MBL is homologous to C1q and triggers the MBLassociated serine proteases (abbr. MASPs), of which the three formsMASP1, MASP2 and MASP3 have been described. Further lectin pathwayactivation is virtually identical to classical pathway activationforming the same C3 and C5 convertases. In addition there is someevidence that MASPs under some conditions may activate C3 directly.

Terminal pathway. All three activation pathways converge in theformation of C5 convertase (C4b2a3b in the classical and lectin pathway,C3bBb3b in the alternative pathway), which cleaves C5 to C5a/b. C5a haspotent anaphylatoxin activity and is chemotactic. The other C5 fragmentC5b functions with its hydrophobic binding site as an anchor on thetarget cell surface to which the lytic membrane attack complex (MAC orterminal complement complex, abbr. TCC) forms. The MAC is assembled fromfive precursor proteins: C5b, C6, C7, C8, and C9. The final event is theformation of C9 oligomers, which insert themselves as transmembranechannels into the plasma membrane leading to osmotic lysis of the cell.MAC assembly is controlled by the soluble plasma factors S protein (alsoknown as vitronectin) and SP-40,40 (also so known as clusterin), and byCD59 and HRF (homologous restriction factor) on host cell membranes.Many kinds of cells are sensitive to complement mediated lysis:erythrocytes, platelets, bacteria, viruses possessing a lipoproteinenvelope, and lymphocytes.

The complement system is a potent mechanism for initiating andamplifying inflammation. This is mediated through fragments of thecomplement components. Anaphylatoxins are the best defined fragments andare proteolytic fragments of the serine proteases of the complementsystem: C3a, C4a and C5a. Anaphylatoxins are not only produced in thecourse of complement activation, but also from activation of otherenzyme systems which may directly cleave C3, C4 and C5. Such enzymesinclude thrombin, plasmin, kallikrein, tissue and leukocyte lysosomalenzymes, and bacterial proteases. The anaphylatoxins have powerfuleffects on blood vessel walls, causing contraction of smooth muscle(e.g. ileal, bronchial, uterine and vascular muscle) and an increase invascular permeability. These effects show specific tachyphylaxis (i.e.repeated stimulation induces diminishing responses) and can be blockedby antihistamines; they are probably mediated indirectly via release ofhistamine from mast cells and basophils. C5a is the 74-amino acidN-terminal cleavage product of the C5 plasmaprotein a chain. It is boundby the receptor C5aR (also known as C5R1 or CD88) with high affinity, amolecule present on many different cell types: most prominently onneutrophils, macrophages, smooth muscle cells, and endothelial cells.C5a is by far the most powerful anaphylatoxin, approximately 100 timesmore effective than C3a, and 1000 times more effective than C4a. Thisactivity decreases in the order C5a>histamine>acetylcholine>C3a>>C4a.

C5a is extremely potent at stimulating neutrophil chemotaxis, adherence,respiratory burst generation and degranulation. C5a also stimulatesneutrophils and endothelial cells to present more adhesion molecules;the intravenous injection of C5a, for example, quickly leads toneutropenia in animal experiments by triggering adherence of neutrophilsto the blood vessel walls. Ligation of the neutrophil C5a receptor isfollowed by mobilization of membrane arachidonic acid which ismetabolized to prostaglandins and leukotrienes including LTB4, anotherpotent chemoattractant for neutrophils and monocytes. Following ligationof monocyte C5a receptors, IL-1 is released. Thus, the local release ofC5a at sites of inflammation results in powerful pro-inflammatorystimuli. In fact, the release of C5a is connected directly or indirectlywith many acute or chronic conditions, such as immune complex associateddiseases in general (Heller et al., 1999); asthma (Kohl, 2001); septicshock (Huber-Lang et al., 2001); systemic inflammtory response syndrome(abbr. SIRS); multiorgan failure (abbr. MOF); acute respiratory distresssyndrome (abbr. ARDS); inflammatory bowel syndrome (abbr. IBD) (Woodruffet al., 2003); infections; severe burns (Piccolo et al., 1999);reperfusion injury of organs such as heart (van der Pals et al. 2010),spleen, bladder, pancreas, stomach, lung, liver, kidney, limbs, brain,sceletal muscle or intestine (Riley et al., 2000); psoriasis (Bergh etal., 1993); myocarditis; multiple sclerosis (Muller-Ladner et al.,1996); and rheumatoid arthritis (abbr.RA) (Woodruff et al., 2002).

Numerous overviews over the relation between the complement system anddiseases are published (Kirschfink, 1997; Kohl, 2001; Makrides, 1998;Walport, 2001a; Walport, 2001b).

Cell injury by complement occurs as a consequence of activation ofeither the classical or the alternative pathway on the surface of acell. The MAC constitutes a supramolecular organisation that is composedof approximately twenty protein molecules and representing a molecularweight of approx. 1.7 million Da. The fully assembled MAC contains onemolecule each of C5b, C6, C7, and C8 and several molecules of C9. Allthese MAC components are glycoproteins. When C5 is cleaved by C5convertase and C5b is produced, self-assembly of the MAC begins. C5b andC6 form a stable and soluble bimolecular complex which binds to C7 andinduces it to express a metastable site through which the nascenttrimolecular complex (C5b-7) can insert itself into membranes, when itoccurs on or in close proximity to a target lipid bilayer. Insertion ismediated by hydrophobic regions on the C5b-7 complex that appearfollowing C7 binding to C5b-6. Membrane-bound C5b-7 commits MAC assemblyto a membrane site and forms the receptor for C8. The binding of one C8molecule to each C5b-7 complex gives rise to small trans-membranechannels of less than 1 nm functional diameter that may perturb targetbacterial and erythrocyte membranes. Each membrane-bound C5b-8 complexacts as a receptor for multiple C9 molecules and appears to facilitateinsertion of C9 into the hydrocarbon core of the cell membrane. Bindingof one molecule of C9 initiates a process of C9 oligomerisation at themembrane attack site. After at least 12 molecules are incorporated intothe complex, a discrete channel structure is formed. Therefore the endproduct consists of the tetramolecular C5b-8 complex (with a molecularweight of approximately 550 kDa) and tubular poly-C9 (with a molecularweight of approximately 1,100 kDa). This form of the MAC, once insertedinto the cell membranes, creates complete transmembrane channels leadingto osmotic lysis of the cell. The transmembrane channels formed vary insize depending on the number of C9 molecules incorporated into thechannel structure. Whereas the presence of poly-C9 is not absolutelyessential for the lysis of red blood cells or of nucleated cells, it maybe necessary for the killing of bacteria.

The complement system is primarily beneficial in the body's defenseagainst invading microorganisms. The early components of the complementcascade are important for opsonization, of infectious agents followed bytheir elimination from the body. In addition, they serve several normalfunctions of the immune system like controlling formation and clearanceof immune complexes or cleaning up debris, dead tissues and foreignsubstances. All three activation pathways which recognize differentmolecular patterns that (in the healthy body) define an extensive arrayof non-self structures help controlling invaders. The terminalcomplement pathway—which culminates in the assembly of theMAC—represents a further line of defense by lysing bacteria and foreigncells.

The importance of a functional complement system becomes clear when theeffects of complement deficiencies are considered. For example,individuals that are missing one of the alternative pathway proteins orlate components (C3-C9) tend to get severe infections with pyogenicorganisms, particularly Neisseria species. Deficiencies in the classicalpathway components (such as C1, C2, C4) are also associated withincreased, though not as strongly elevated, risk of infection.Complement components like C1 and MBL do also have the ability toneutralize viruses by interfering with the viral interaction with thehost cell membrane, thus preventing entrance into the cell.

Of note, although cleavage of C5 leads to C5a as well as the MAC, theclinical features of C5 deficiency do not differ markedly from those ofother terminal component deficiencies (e.g. C6, C7, C8, C9) suggestingthat the absence of C5a does not contribute significantly to theclinical picture in C5-deficient patients. Therefore, the selectiveantagonisation of C5a promises to be the optimal leverage, so that thenormal up- and downstream disease-preventing functions of complementremain intact. Thus, only the deleterious overproduction of theproinflammatory anaphylatoxin is blocked.

The fact that C5aR-deficient mice—although they are more susceptible forinfections with Pseudomonas aeruginosa—appear otherwise normal, suggeststhat the blockade of C5a function does not have deleterious effects.

Several compounds targeting C5a or C5 or the respective receptor areknown and were successfully tested in in vivo models. Some of them havebeen further tested in clinical trials. The C5-specific humanizedantibody, eculizumab is approved for paroxysmal nocturnal hemoglobinuriaand has shown efficacy in treating atypical haemolytic uraemic syndrome(aHUS), acute antibody-mediated kidney allograft rejection and coldagglutinin disease. It prevents cleavage of C5 and inhibits the actionof both C5a and C5b. Besides similar research-stage C5 antibodies andantibody fragments, antibodies that selectively disrupt C5a:C5aR (CD88)interaction and leaves C5 cleavage and C5b-dependent MAC-formationunaffected are of special interest. Examples are the humanized anti-C5amAb MEDI-7814 that is in Phase I clinical development for the potentialiv treatment of inflammatory disorders and tissue injury and the C5aantibody TNX-558 for which however no development has been reportedsince 2007. An antibody to the C5a receptor, neutrazumab, is underdevelopment for rheumatoid arthritis and stroke (Ricklin & Lambris 2007;Wagner & Frank 2010).

A PEGylated anti-C5 aptamer (ARC-1905) is in preclinical development forAMD. CCX168 is a small molecule C5aR inhibitor currently in Phase IIclinical development for anti-neutrophil cytoplasmicautoantibody-associated vasculitides (ChemoCentryx Press Release Oct.17, 2011). Another C5aR antagonist in clinical development is MP-435 forthe treatment of rheumatoid arthritis.

No development has been reported for the small molecule/peptidomimeticC5a receptor antagonists JPE-1375, JSM-7717 recently (Ricklin & Lambris2007). Another inhibitor of the C5a receptor CD88, the cyclichexapeptide PMX53, has been efficacious in inflammatory animal models,but has not met endpoints in placebo-controlled double-blind clinicalstudies in patients with rheumatoid arthritis. The clinical developmentfor AMD has also been discontinued (Wagner & Frank 2010). Aresearch-stage variant of PMX53, PMX205, has been published to be ativein a murine model of Alzheimer's dementia (Fonseca et al. 2009). Afurther clinical stage compound is the C5a receptor (C5aR) antagonist,CCX-168. A Phase I trial has been initiated initiated for inflammatoryand autoimmune diseases in January 2010.

Beside the effects of C5a as described supra, new data let assume thatthe generation of C5a in a tumor microenviroment enhance tumor growth bythe suppression the antitumor CD8+ T-cell-mediated response, wherebysaid suppression seems to be associated with the recruitement ofmyeloid-derived suppressor cells into tumors and augmentation of theirT-cell-directed suppressive abilities. Markiewski et al. showed that ablockade of the C5a receptors by a pepdidic C5a receptor antagonist ledto a retarded tumor growth in a mouse model (Markiewski et al., 2008).

Most of peptidic compounds are prone to degradation and modification bypeptidases and additionally show a fast clearance rate from the body,preferably the human body. Thus, these peptidic compounds cannot beconsidered as drug-like molecules, a prerequisite for the development ofdrugs in general to be marketed.

Several Spiegelmers specifically binding to human C5a, but not to C5a ofother species, were developed in the past (see WO2009/040113 andWO2010/108657).

Because for pre-clinical and clinical development animal models areessential, the problem underlying the present invention is to provide acompound which interacts with mouse C5a. More specifically, the problemunderlying the present invention is to provide for a compound whichinteracts with both mouse C5a and human C5a.

A further problem underlying the present invention is to provide acompound for the manufacture of a medicament for the treatment of ahuman, and/or non-human diseases, whereby the disease is characterizedby C5a being either directly or indirectly involved in the pathogeneticmechanism of such disease.

A still further problem underlying the present invention is to provide acompound for the manufacture of a diagnostic agent for the treatment ofa disease, whereby the disease is characterized by C5a being eitherdirectly or indirectly involved in the pathogenetic mechanism of suchdisease.

These and other problems underlying the present invention are solved bythe subject matter of the attached independent claims. Preferredembodiments may be taken from the dependent claims.

The problem underlying the present invention is solved in a firstaspect, which is also the first embodiment of the first aspect, by anucleic acid molecule capable of binding to human C5a, wherein thenucleic acid molecule comprises a central stretch of nucleotides,wherein the central stretch of nucleotides comprises a nucleotidesequence of

[SEQ ID NO: 61] 5′ AUGn₁GGUGKUn₂n₃RGGGHUGUKGGGn₄Gn₅CGACGCA 3′,

wherein

n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅ is U ordU and

G, A, U, C, H, K, and R are ribonucleotides, and

dU, dG and dA are 2′-deoxyribonucleotides.

In a second embodiment of the first aspect which is also an embodimentof the first embodiment of the first aspect, the central stretch ofnucleotides comprises a nucleotide sequence selected from the group of

[SEQ ID NO: 62] a) 5′ AUGn₁GGUGUUn₂n₃AGGGUUGUGGGGn₄Gn₅CGACGCA 3′,[SEQ ID NO: 63] b) 5′ AUGn₁GGUGUUn₂n₃GGGGUUGUGGGGn₄Gn₅CGACGCA 3′,[SEQ ID NO: 64] c) 5′ AUGn₁GGUGUUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA 3′,[SEQ ID NO: 65] d) 5′ AUGn₁GGUGGUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA 3′,[SEQ ID NO: 66] e) 5′ AUGn₁GGUGGUn₂n₃GGGGUUGUGGGGn₄Gn₅CGACGCA 3′,[SEQ ID NO: 67] f) 5′ AUGn₁GGUGGUn₂n₃GGGGAUGUGGGGn₄Gn₅CGACGCA 3′, and[SEQ ID NO: 68] g) 5′ AUGn₁GGUGUUn₂n₃GGGGCUGUGGGGn₄Gn₅CGACGCA 3′,

wherein

n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅ is U ordU and

G, A, U and C are ribonucleotides, and

dU, dG and dA are 2′-deoxyribonucleotides.

In a third embodiment of the first aspect which is also an embodiment ofthe second embodiment of the first aspect, the central stretch ofnucleotides comprises a nucleotide sequence of

[SEQ ID NO: 65] 5′ AUGn₁GGUGGUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA 3′

wherein

n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅ is U ordU and

G, A, U and C are ribonucleotides, and

dU, dG and dA are 2′-deoxyribonucleotides.

In a fourth embodiment of the first aspect which is also an embodimentof the third embodiment of the first aspect, the central stretch ofnucleotides comprises a nucleotide sequence selected from the group of

[SEQ ID NO: 73] a) 5′ AUGdUGGUGGUGAAGGGUUGUUGGGUGUCGACGCA 3′,[SEQ ID NO: 74] b) 5′ AUGUGGUGGUdGAAGGGUUGUUGGGUGUCGACGCA 3′,[SEQ ID NO: 75] c) 5′ AUGUGGUGGUGdAAGGGUUGUUGGGUGUCGACGCA 3′,[SEQ ID NO: 76] d) 5′ AUGUGGUGGUGAAGGGUUGUUGGGdUGUCGACGCA 3′,[SEQ ID NO: 77] e) 5′ AUGUGGUGGUGAAGGGUUGUUGGGUGdUCGACGCA 3′,[SEQ ID NO: 78] f) 5′ AUGdUGGUGGUGAAGGGUUGUUGGGdUGUCGACGCA 3′,[SEQ ID NO: 79] g) 5′ AUGdUGGUGGUGAAGGGUUGUUGGGUGdUCGACGCA 3′,[SEQ ID NO: 80] h) 5′ AUGUGGUGGUGAAGGGUUGUUGGGdUGdUCGACGCA 3′,[SEQ ID NO: 81] i) 5′ AUGdUGGUGGUGAAGGGUUGUUGGGdUGdUCGACGCA 3′,[SEQ ID NO: 82] j) 5' AUGdUGGUGGUdGAAGGGUUGUUGGGdUGdUCGACGCA 3'[SEQ ID NO: 83] k) 5′ AUGdUGGUGGUGdAAGGGUUGUUGGGdUGdUCGACGCA 3′,[SEQ ID NO: 84] l) 5′ AUGdUGGUGGUdGdAAGGGUUGUUGGGdUGdUCGACGCA 3′,

preferably the central stretch of nucleotides is

[SEQ ID NO: 78] 5′ AUGdUGGUGGUGAAGGGUUGUUGGGdUGUCGACGCA 3′ or[SEQ ID NO: 84] 5′ AUGdUGGUGGUdGdAAGGGUUGUUGGGdUGdUCGACGCA 3′.

In a fifth embodiment of the first aspect which is also an embodiment ofthe second embodiment of the first aspect, the central stretch ofnucleotides comprises a nucleotide sequence of

[SEQ ID NO: 66] 5′ AUGn₁GGUGGUn₂n₃GGGGUUGUGGGGn₄Gn₅CGACGCA 3′,

wherein

n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅ is U ordU and

G, A, U and C are ribonucleotides, and

dU, dG and dA are 2′-deoxyribonucleotides.

In a sixth embodiment of the first aspect which is also an embodiment ofthe second embodiment of the first aspect, the central stretch ofnucleotides comprises a nucleotide sequence of

[SEQ ID NO: 67] 5′ AUGn₁GGUGGUn₂n₃GGGGAUGUGGGGn₄Gn₅CGACGCA 3′,

wherein

n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅ is U ordU and

G, A, U and C are ribonucleotides, and

dU, dG and dA are 2′-deoxyribonucleotides.

In a seventh embodiment of the first aspect which is also an embodimentof the second embodiment of the first aspect, the central stretch ofnucleotides comprises a nucleotide sequence of

[SEQ ID NO: 64] 5′ AUGn₁GGUGUUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA 3′,

wherein

n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅ is U ordU and

G, A, U and C are ribonucleotides, and

dU, dG and dA are 2′-deoxyribonucleotides.

In an eighth embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth and seventh embodimentof the first aspect, the central stretch of nucleotides consists ofribonucleotides and 2′-deoxyribonucleotides.

In a ninth embodiment of the first aspect which is also an embodiment ofthe first, second, third, fifth, sixth and seventh embodiment of thefirst aspect, the central stretch of nucleotides consists ofribonucleotides.

In a tenth embodiment of the first aspect which is also an embodiment ofthe first, second, third, fourth, fifth, sixth, seventh, eighth andninth embodiment of the first aspect, the nucleic acid moleculecomprises in 5′→3′ direction a first terminal stretch of nucleotides,the central stretch of nucleotides and a second terminal stretch ofnucleotides, wherein

-   -   the first terminal stretch of nucleotides comprises one to five        nucleotides, and    -   the second terminal stretch of nucleotides comprises one to five        nucleotides, preferably    -   the first terminal stretch of nucleotides comprises three to        five nucleotides, and    -   the second terminal stretch of nucleotides comprises three to        five nucleotides, more preferably    -   the first terminal stretch of nucleotides comprises three        nucleotides, and    -   the second terminal stretch of nucleotides comprises three        nucleotides.

In an eleventh embodiment of the first aspect which is also anembodiment of the tenth embodiment of the first aspect, the firstterminal stretch of nucleotides comprises a nucleotide sequence of 5′Z₁Z₂Z₃Z₄G 3′ and the second terminal stretch of nucleotides comprises anucleotide sequence of 5′ Z₅Z₆Z₇Z₈Z₉ 3′,

wherein

Z₁ is G or absent, Z₂ is S or absent, Z₃ is S or absent, Z₄ is B orabsent, Z₅ is C or dC, Z₆ is V or absent, Z₇ is S or absent, Z₈ is S orabsent, Z₉ is C or absent, and

G, S, B, C, V are ribonucleotides, and

dC is a 2′-deoxyribonucleotide,

preferably

-   -   a) Z₁ is G, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is V,        Z₇ is S, Z₈ is S, Z₉ is C, or    -   b) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   c) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   d) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₁ is absent, Z₈ is absent, Z₉ is absent, or    -   e) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   f) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   g) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   h) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is absent, Z₅ is        C or dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   i) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   j) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   k) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is absent, Z₅ is        C or dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   l) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   m) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is absent, Z₈ is absent, Z₉ is absent, or    -   n) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is absent, Z₅ is        C, Z₆ is V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   o) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   p) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is absent, Z₈ is absent, Z₉ is absent, or    -   q) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is absent, Z₇ is absent, Z₈ is absent, Z₉ is absent.

In a twelfth embodiment of the first aspect which is also an embodimentof the tenth and eleventh embodiment of the first aspect, the firstterminal stretch of nucleotides comprises a nucleotide sequence of 5′GCCUG 3′ and the second terminal stretch of nucleotides comprises anucleotide sequence of 5′ CAGGC 3′ or of 5′ dCAGGC 3′, wherein

C, A, G and U are ribonucleotides, and

dC is a 2′-deoxyribonucleotide.

In a 13^(th) embodiment of the first aspect which is also an embodimentof the twelfth embodiment of the first aspect, the second terminalstretch of nucleotides comprises a nucleotide sequence of 5′ dCAGGC 3′.

In a 14^(th) embodiment of the first aspect which is also an embodimentof the twelfth embodiment of the first aspect, the second terminalstretch of nucleotides comprises a nucleotide sequence of 5′ CAGGC 3′.

In a 15^(th) embodiment of the first aspect which is also an embodimentof the tenth and eleventh embodiment of the first aspect, the the firstterminal stretch of nucleotides comprises a nucleotide sequence of 5′CCUG 3′ or 5′ CUG 3′ or 5′ UG 3′ or 5′ G 3′, and the second terminalstretch of nucleotides comprises a nucleotide sequence of 5′ dCAGGC 3′,wherein

C, A, G and U are ribonucleotides, and

dC is a 2′-deoxyribonucleotide.

In a 16^(th) embodiment of the first aspect which is also an embodimentof the 15^(th) embodiment of the first aspect, the

the first terminal stretch of nucleotides comprises a nucleotidesequence of 5′ CCUG 3′.

In a 17^(th) embodiment of the first aspect which is also an embodimentof the 15^(th) embodiment of the first aspect, the

the first terminal stretch of nucleotides comprises a nucleotidesequence of 5′ CUG 3′.

In an 18^(th) embodiment of the first aspect which is also an embodimentof the tenth and eleventh embodiment of the first aspect,

-   a) the first terminal stretch of nucleotides comprises a nucleotide    sequence of 5′ GCUG 3′ and the second terminal stretch of    nucleotides comprises a nucleotide sequence of 5′ dCAGC 3′; or-   b) the first terminal stretch of nucleotides comprises a nucleotide    sequence of 5′ GCCG 3′ and the second terminal stretch of    nucleotides comprises a nucleotide sequence of 5′ dCGGC 3′; or-   c) the first terminal stretch of nucleotides comprises a nucleotide    sequence of 5′ GGCG 3′ and the second terminal stretch of    nucleotides comprises a nucleotide sequence of 5′ dCGCC 3′; wherein

C, A, G and U are ribonucleotides, and

dC is a 2′-deoxyribonucleotide.

In a 19^(th) embodiment of the first aspect which is also an embodimentof the 18^(th) embodiment of the first aspect, first terminal stretch ofnucleotides comprises a nucleotide sequence of 5′ GGCG 3′ and the secondterminal stretch of nucleotides comprises a nucleotide sequence of 5′dCGCC 3′.

In a 20^(th) embodiment of the first aspect which is also an embodimentof the tenth and eleventh embodiment of the first aspect, the

the first terminal stretch of nucleotides comprises a nucleotidesequence of 5′ CUG 3′ or 5′ UG 3′ or 5′ CG 3′ or 5′ G 3′, and

the second terminal stretch of nucleotides comprises a nucleotidesequence of 5′ dCAGC 3′, wherein

C, A, G and U are ribonucleotides, and

dC is a 2′-deoxyribonucleotide.

In a 21^(st) embodiment of the first aspect which is also an embodimentof the tenth and eleventh embodiment of the first aspect,

the first terminal stretch of nucleotides comprises a nucleotidesequence of 5′ GCUG 3′, and

the second terminal stretch of nucleotides comprises a nucleotidesequence of 5′ dCAC 3′ or 5′ dCC 3′ or 5′ dCA 3′, wherein

C, A, G and U are ribonucleotides, and

dC is a 2′-deoxyribonucleotide.

In a 22^(nd) embodiment of the first aspect which is also an embodimentof the tenth and eleventh embodiment of the first aspect,

-   -   a) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GUG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCAC 3′; or    -   b) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ UG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCA 3′; or    -   c) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCGC 3′; or    -   d) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ CG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCGC 3′; or    -   e) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ G 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCGC 3′; or    -   f) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCC 3′; or    -   g) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dC 3′; or    -   h) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCC 3′;    -   i) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ CGC 3′;        wherein

C, A, U and G are ribonucleotides, and

dC is a 2′-deoxyribonucleotide.

In a 23^(rd) embodiment of the first aspect which is also an embodimentof the 22^(nd) embodiment of the first aspect, the first terminalstretch of nucleotides comprises a nucleotide sequence of 5′ GCG 3′ andthe second terminal stretch of nucleotides comprises a nucleotidesequence of 5′ dCGC 3′.

In a 24^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fifth, sixth, seventh, ninth, tenth,eleventh, twelfth and 14^(th) embodiment of the first aspect, thenucleic acid molecule comprises a nucleotide sequence selected from thegroup of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQID NO: 90, or a nucleic acid molecule having an identity of at least 85%to the nucleic acid molecule comprising a nucleotide sequence selectedfrom the group of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6and SEQ ID NO: 90, or a nucleic acid molecule which is homologous to thenucleic acid molecule comprising a nucleotide sequence selected from thegroup of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQID NO: 90, wherein the homology is at least 85%.

In a 25^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, eighth, tenth, eleventh, twelfth,13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th),21^(st), 22^(nd) and 23^(rd) embodiment of the first aspect, the nucleicacid molecule comprises a nucleotide sequence selected from the group ofSEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 91 and SEQ ID NO: 92, or a nucleic acid moleculehaving an identity of at least 85% to the nucleic acid moleculecomprising a nucleotide sequence selected from the group of 14, SEQ IDNO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQID NO: 37, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 91and SEQ ID NO: 92, or a nucleic acid molecule which is homologous to thenucleic acid molecule comprising a nucleotide sequence selected from thegroup of 14, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 91 and SEQ ID NO: 92, wherein the homology is at least85%.

In a 26^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th)and 25^(th) embodiment of the first aspect, the nucleic acid molecule iscapable of binding human C5a and mouse C5a.

In a 27^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th) and 26^(th) embodiment of the first aspect, the nucleic acidmolecule comprises at least one binding moiety which is capable ofbinding human C5a and mouse C5a, wherein such binding moiety consists ofL-nucleotides.

In a 28^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th), 26^(th) and 27^(th) embodiment of the first aspect, thenucleotides of or the nucleotides forming the nucleic acid molecule areL-nucleotides.

In a 29^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th), 26^(th), 27^(th) and 28^(th) embodiment of the first aspect,the nucleic acid molecule is an L-nucleic acid molecule.

In a 30^(th) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th), 26^(th), 27^(th), 28^(th) and 29^(th) embodiment of the firstaspect, the nucleic acid is an antagonist of an activity mediated byhuman and/or mouse C5a.

In a 31^(st) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th), 26^(th), 27^(th), 28^(th), 29^(th) and 30^(th) embodiment ofthe first aspect, the nucleic acid molecule comprises a modificationgroup, wherein excretion rate of the nucleic acid molecule comprisingthe modification group from an organism is decreased compared to anucleic acid not comprising the modification group.

In a 32^(nd) embodiment of the first aspect which is also an embodimentof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th), 26^(th), 27^(th), 28^(th), 29^(th) and 30^(th) embodiment ofthe first aspect, the nucleic acid molecule comprises a modificationgroup, wherein the nucleic acid molecule comprising the modificationgroup has an increased retention time in an organism compared to anucleic acid molecule not comprising the modification group.

In a 33^(rd) embodiment of the first aspect which is also an embodimentof the 31^(st) and 32^(rd) embodiment of the first aspect, themodification group is selected from the group comprising biodegradableand non-biodegradable modifications, preferably the modification groupis selected from the group comprising polyethylene glycol, linearpolyethylene glycol, branched polyethylene glycol, hydroxyethyl starch,a peptide, a protein, a polysaccharide, a sterol, polyoxypropylene,polyoxyamidate and poly (2-hydroxyethyl)-L-glutamine.

In a 34^(th) embodiment of the first aspect which is also an embodimentof the 33^(rd) embodiment of the first aspect, the modification group isa polyethylene glycol, preferably consisting of a linear polyethyleneglycol or branched polyethylene glycol, wherein the molecular weight ofthe polyethylene glycol is preferably from about 20,000 to about 120,000Da, more preferably from about 30,000 to about 80,000 Da and mostpreferably about 40,000 Da.

In a 35^(th) embodiment of the first aspect which is also an embodimentof the 33^(rd) embodiment of the first aspect, the modification group ishydroxyethyl starch, wherein preferably the molecular weight of thehydroxyethyl starch is from about 50 to about 1000 kDa, more preferablyfrom about 100 to about 700 kDa and most preferably from 200 to 500 kDa.

In a 36^(th) embodiment of the first aspect which is also an embodimentof the 31^(st), 32^(nd), 33^(rd), 34^(th) and 35^(th) embodiment of thefirst aspect, the modification group is coupled to the nucleic acidmolecule via a linker, whereby preferably the linker is a biodegradablelinker.

In a 37^(th) embodiment of the first aspect which is also an embodimentof the 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th) and 36^(th)embodiment of the first aspect, the modification group is coupled to the5′-terminal nucleotide and/or the 3′-terminal nucleotide of the nucleicacid molecule and/or to a nucleotide of the nucleic acid moleculebetween the 5′-terminal nucleotide of the nucleic acid molecule and the3′-terminal nucleotide of the nucleic acid molecule.

In a 38^(th) embodiment of the first aspect which is also an embodimentof the 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th) and 37^(th)embodiment of the first aspect, the organism is an animal or a humanbody, preferably a human body.

The problem underlying the present invention is solved in a secondaspect, which is also the first embodiment of the second aspect, by anucleic acid molecule according to the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, 13^(th),14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th), 21^(st),22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th), 27^(th), 28^(th), 29^(th),30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th), 37^(th)and 38^(th) embodiment of the first aspect, for use in a method for thetreatment and/or prevention of a disease.

In a second embodiment of the second aspect which is also an embodimentof the first embodiment of the second aspect, the disease is associatedwith complement activation and C5a-mediated pathogenic mechanisms,and/or is selected from the group comprising of autoimmune disease,inflammatory disease, systemic inflammatory response syndrome, diseaseof the eye, ischemia/reperfusion injuries, delayed graft function,transplant rejection, cardiovascular disease, respiratory disease, acutereactions, infectious disease, neurological disease, neurodegenerativedisease, fibrotic disease, hematological disease, metabolic disease,tumors and clinical complications associated with complement activationby biomaterials, preferably the systemic inflammatory response syndromeis selected from the group comprising sepsis and secondary damages oftrauma or severe burns.

The problem underlying the present invention is solved in a thirdaspect, which is also the first embodiment of the third aspect, by apharmaceutical composition comprising a nucleic acid molecule accordingto the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th),25^(th), 26^(th), 27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd),33^(rd), 34^(th), 35^(th), 36^(th), 37th and 38th embodiment of thefirst aspect and according to the first embodiment of the second aspect,and optionally a further constituent, wherein the further constituent isselected from the group comprising a pharmaceutically acceptableexcipient, a pharmaceutically acceptable carrier and a pharmaceuticallyactive agent.

In a second embodiment of the third aspect which is also an embodimentof the first embodiment of the third aspect, the pharmaceuticalcomposition comprises a nucleic acid molecule according to the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th),35^(th), 36^(th), 37th and 38th embodiment of the first aspect andaccording to the first embodiment of the second aspect, and apharmaceutically acceptable carrier.

The problem underlying the present invention is solved in a fourthaspect, which is also the first embodiment of the fourth aspect, by theuse of a nucleic acid molecule according to the first, second, third,fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th),21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th), 27^(th), 28^(th),29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th),37th and 38th embodiment of the first aspect and according to the firstembodiment of the second aspect, for the manufacture of a medicament.

In a second embodiment of the fourth aspect which is also an embodimentof the first embodiment of the fourth aspect, the medicament is for usein human medicine or for use in veterinary medicine.

In a third embodiment of the fourth aspect which is also an embodimentof the first and the second embodiment of the fourth aspect, themedicament is for the treatment and/or prevention of autoimmune disease,inflammatory disease, systemic inflammatory response syndrome, diseaseof the eye, ischemia/reperfusion injuries, delayed graft function,transplant rejection, cardiovascular disease, respiratory disease, acutereactions, infectious disease, neurological disease, neurodegenerativedisease, fibrotic disease, hematological disease, metabolic disease,tumors and clinical complications associated with complement activationby biomaterials, preferably the systemic inflammatory response syndromeis selected from the group comprising sepsis and secondary damages oftrauma or severe burns.

The problem underlying the present invention is solved in a fifthaspect, which is also the first embodiment of the fifth aspect, by theuse of a nucleic acid molecule according to the first, second, third,fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th),21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th), 27^(th), 28^(th),29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th),37th and 38th embodiment of the first aspect and according to the firstembodiment of the second aspect, for the manufacture of a diagnosticmeans.

The problem underlying the present invention is solved in a sixthaspect, which is also the first embodiment of the sixth aspect, by acomplex comprising a nucleic acid molecule according to the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th),35^(th), 36^(th), 37th and 38th embodiment of the first aspect andaccording to the first embodiment of the second aspect, and C5a, whereinpreferably the complex is a crystalline complex.

The problem underlying the present invention is solved in a seventhaspect, which is also the first embodiment of the seventh aspect, by theuse of a nucleic acid molecule according to the first, second, third,fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th),21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th), 27^(th), 28^(th),29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th),37th and 38th embodiment of the first aspect and according to the firstembodiment of the second aspect, for the detection of C5a.

The problem underlying the present invention is solved in an eighthaspect, which is also the first embodiment of the eighth aspect, by amethod for the screening of an antagonist of an activity mediated by C5acomprising the following steps:

-   -   providing a candidate antagonist of the activity mediated by        C5a,    -   providing a nucleic acid molecule according to the first,        second, third, fourth, fifth, sixth, seventh, eighth, ninth,        tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),        17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd),        24^(th), 25^(th), 26^(th), 27^(th), 28^(th), 29^(th), 30^(th),        31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th), 37th and        38th embodiment of the first aspect and according to the first        embodiment of the second aspect,    -   providing a test system which provides a signal in the presence        of an antagonist of the activity mediated by C5a, and    -   determining whether the candidate antagonist of the activity        mediated by C5a is an antagonist of the activity mediated by        C5a.

The problem underlying the present invention is solved in a ninthaspect, which is also the first embodiment of the ninth aspect, by a kitfor the detection of C5a, wherein the kit comprises a nucleic acidmolecule according to the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, 13^(th), 14^(th),15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd),23^(rd), 24^(th), 25^(th), 26^(th), 27^(th), 28^(th), 29^(th), 30^(th),31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th), 37th and 38thembodiment of the first aspect and according to the first embodiment ofthe second aspect, and at least an instruction leaflet or a reactionvessel.

The problem underlying the present invention is solved in a tenthaspect, which is also the first embodiment of the tenth aspect, by amethod for the detection of a nucleic acid according to the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th),35^(th), 36^(th), 37th and 38th embodiment of the first aspect andaccording to the first embodiment of the second aspect, in a sample,wherein the method comprises the steps of:

-   -   a) providing a capture probe, wherein the capture probe is at        least partially complementary to a first part of the nucleic        acid molecule according to the first, second, third, fourth,        fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,        13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th),        20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),        27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd),        34^(th), 35^(th), 36^(th), 37th and 38th embodiment of the first        aspect and according to the first embodiment of the second        aspect, and a detection probe, wherein the detection probe is at        least partially complementary to a second part of the nucleic        acid molecule according to the first, second, third, fourth,        fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,        13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th),        20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),        27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd),        34^(th), 35^(th), 36^(th), 37th and 38th embodiment of the first        aspect and according to the first embodiment of the second        aspect, or, alternatively, the capture probe is at least        partially complementary to a second part of the nucleic acid        molecule according to the first, second, third, fourth, fifth,        sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,        13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th),        20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),        27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd),        34^(th), 35^(th), 36^(th), 37th and 38th embodiment of the first        aspect and according to the first embodiment of the second        aspect, and the detection probe is at least partially        complementary to a first part of the nucleic acid molecule        according to the first, second, third, fourth, fifth, sixth,        seventh, eighth, ninth, tenth, eleventh, twelfth, 13^(th),        14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th),        21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th), 27^(th),        28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th),        35^(th), 36^(th), 37th and 38th embodiment of the first aspect        and according to the first embodiment of the second aspect;    -   b) adding the capture probe and the detection probe separately        or combined to a sample containing the nucleic acid molecule        according to the first, second, third, fourth, fifth, sixth,        seventh, eighth, ninth, tenth, eleventh, twelfth, 13^(th),        14^(th), 15^(th), 16^(th), 17^(th), 18^(th), 19^(th), 20^(th),        21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th), 27^(th),        28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th),        35^(th), 36^(th), 37th and 38th embodiment of the first aspect        and according to the first embodiment of the second aspect, or        presumed to contain the nucleic acid molecule according to the        first, second, third, fourth, fifth, sixth, seventh, eighth,        ninth, tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th),        16^(th), 17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd),        23^(rd), 24^(th), 25^(th), 26^(th), 27^(th), 28^(th), 29^(th),        30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th),        37th and 38th embodiment of the first aspect and according to        the first embodiment of the second aspect;    -   c) allowing the capture probe and the detection probe to react        either simultaneously or in any order sequentially with the        nucleic acid molecule according to the first, second, third,        fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,        twelfth, 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),        19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th),        26^(th), 27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd),        33^(rd), 34^(th), 35^(th), 36^(th), 37th and 38th embodiment of        the first aspect and according to the first embodiment of the        second aspect, or part thereof;    -   d) optionally detecting whether or not the capture probe is        hybridized to the nucleic acid molecule according to the first,        second, third, fourth, fifth, sixth, seventh, eighth, ninth,        tenth, eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th),        17^(th), 18^(th), 19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd),        24^(th), 25^(th), 26^(th), 27^(th), 28^(th), 29^(th), 30^(th),        31^(st), 32^(nd), 33^(rd), 34^(th), 35^(th), 36^(th), 37th and        38th embodiment of the first aspect and according to the first        embodiment of the second aspect, provided in step a); and    -   e) detecting the complex formed in step c) consisting of the        nucleic acid molecule according to the first, second, third,        fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,        twelfth, 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),        19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th),        26^(th), 27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd),        33^(rd), 34^(th), 35^(th), 36^(th), 37th and 38th embodiment of        the first aspect and according to the first embodiment of the        second aspect, and the capture probe and the detection probe.

In a second embodiment of the tenth aspect which is also an embodimentof the first embodiment of the tenth aspect, the detection probecomprises a detection means, and/or wherein the capture probe isimmobilized to a support, preferably a solid support.

In a third embodiment of the tenth aspect which is also an embodiment ofthe first and the second embodiment of the tenth aspect, any detectionprobe which is not part of the complex formed in step c) is removed fromthe reaction so that in step e) only a detection probe which is part ofthe complex, is detected.

In a fourth embodiment of the tenth aspect which is also an embodimentof the first, second and third embodiment of the tenth aspect, step e)comprises the step of comparing the signal generated by the detectionmeans when the capture probe and the detection probe are hybridized inthe presence of the nucleic acid molecule according to the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), 18^(th),19^(th), 20^(th), 21^(st), 22^(nd), 23^(rd), 24^(th), 25^(th), 26^(th),27^(th), 28^(th), 29^(th), 30^(th), 31^(st), 32^(nd), 33^(rd), 34^(th),35^(th), 36^(th), 37th and 38th embodiment of the first aspect andaccording to the first embodiment of the second aspect, or part thereof,and in the absence of said.

While not wishing to be bound by any theory, the present inventors havesurprising found that the nucleic acid molecule according to the presentinvention binds specifically and with high affinity to both mouse C5aand human C5a, thereby inhibiting the binding of C5a to its C5areceptor, although the sequence homology of mouse C5a and human C5a isonly 64% in the homologous region and mouse C5a having 3 additionalN-terminal amino acids. Moreover, in contrast to human C5a, mouse C5a isnot glycosylated. Asparagine⁶⁴, the glycosylation site in human C5a ismutated to glutamate in mouse. In particular the shown affinity ofseveral individual C5a binding nucleic acids in the picomolar rangecould not be foreseen.

Furthermore, the instant inventors have surprisingly found that thenucleic acid molecule according to the present invention is suitable toblock the interaction of C5a with the C5a receptor. Insofar, the nucleicacid molecule according to the present invention can also be viewed asan antagonist of the C5a receptor and, respectively, as an antagonist ofthe effects of C5a, in particular the effects of C5a on its receptor. Anantagonist to C5a is a molecule that binds to C5a—such as the nucleicacid molecules according to the present invention—and inhibts thefunction of C5a, preferably in an in vitro assay or in an in vivo modelas described in the Examples.

It is within the present invention that the nucleic acid according tothe present invention is a nucleic acid molecule. Insofar the termsnucleic acid and nucleic acid molecule are used herein in a synonymousmanner if not indicated to the contrary. Moreover, such nucleic acid(s)is/are preferably also referred to herein as the nucleic acidmolecule(s) according to the (present) invention, the nucleic acid(s)according to the present invention, the inventive nucleic acid(s) or theinventive nucleic acid molecule(s).

The features of the nucleic acids according to the present invention asdescribed herein can be realised in any aspect of the present inventionwhere the nucleic acid is used, either alone or in any combination.

As to the various diseases, conditions and disorders which may betreated or prevented by using the nucleic acid molecule according to thepresent invention and compositions, preferably pharmaceuticalcompositions comprising the same, it has to be acknowledged that suchdiseases, conditions and disorders are those which are described herein,including and in particular those described and set forth in theintroductory part of the instant application. Insofar, the respectivepassages of the specification and the introductory part of thespecification form an integral part of the present disclosure teachingthe suitability of the nucleic acid molecule of the present inventionfor the prevention and treatment, respectively, for said diseases,conditions, and disorders. Additionally, a nucleic molecule according tothe present invention is preferred if the physiological effect of theC5a-C5a receptor axis is related to higher plasma levels of C5a.

As used herein the term C5a refers to any C5a including, but not limitedto, mammalian C5a. Preferably, the mammalian C5a is selected from thegroup comprising human, rat, mouse, monkey C5a (see C5a speciesalignment in FIG. 11). More preferably the C5a is human C5a. Human C5ais a basic protein having the amino acid sequence according to SEQ. ID.No. 50. Mouse C5a is a basic protein having the amino acid sequenceaccording to SEQ. ID. No. 52.

As outlined in more detail in the claims and example 1, the presentinventors could more surprisingly identify a number of different bindingnucleic acid molecules capable of binding both human and mouse C5a.

As outlined in more detail herein, the present inventors have identifieda number of different C5a binding nucleic acid molecules capable ofbinding both, human and mouse C5a, whereby the nucleic acid moleculescan be characterised in terms of stretches of nucleotides which are alsoreferred to herein as disclosed (see Example 1). As experimentally shownin examples 8 and 9 the inventors could surprisingly demonstrate inseveral systems that the nucleic acid molecule according to the presentinvention is suitbale for the treatment of sepsis.

Each of the different types of C5a binding nucleic acid molecules of theinvention that bind to C5a comprises three different stretches ofnucleotides: a first terminal stretch of nucleotides, a central stretchof nucleotides and a second terminal stretch of nucleotides. In general,C5a binding nucleic acid molecules of the present invention comprise attheir 5′-end and the 3′-end each one of the terminal stretches ofnucleotides, i.e. the first terminal stretch of nucleotides or thesecond terminal stretch of nucleotides (also referred to as 5′-terminalstretch of nucleotides and 3′-terminal stretch of nucleotides). Thefirst terminal stretch of nucleotides and the second terminal stretch ofnucleotides can, in principle due to their base complementarity,hybridize to each other, whereby upon hybridization a double-strandedstructure is formed. However, such hybridization is not necessarilyrealized in the molecule under physiological and/or non-physiologicalconditions. The three stretches of nucleotides of C5a binding nucleicacid molecules—the first terminal stretch of nucleotides, the centralstretch of nucleotides and second terminal stretch of nucleotides—arearranged to each other in 5′→3′-direction: the first terminal stretch ofnucleotides—the central stretch of nucleotides—the second terminalstretch of nucleotides. Alternatively, the second terminal stretch ofnucleotides, the central stretch of nucleotides and the terminal firststretch of nucleotides are arranged to each other in 5′→3′-direction.

The length of the central stretch of nucleotides of the nucleic acidsaccording to the present invention is preferably 34.

The length of the first terminal stretch of nucleotides of the nucleicacids according to the present invention is between one and fivenucleotides, preferably between three and five nucleotides, morepreferably three nucleotides.

The length of the second terminal stretch of nucleotides of the nucleicaccording to the present invention is between one and five nucleotides,preferably between three and five nucleotides, more preferably threenucleotides.

The terms ‘stretch’ and ‘stretch of nucleotides’ are used herein in asynonymous manner if not indicated to the contrary.

The differences in the sequences of the defined stretches between thedifferent C5a binding nucleic acid molecules may influence the bindingaffinity to C5a. Based on binding analysis of the different C5a bindingnucleic acid molecules of the present invention the central stretch andthe nucleotides forming the same are individually and more preferably intheir entirety essential for binding of the C5a binding nucleic acidmolecule to C5a.

In a preferred embodiment the nucleic acid molecule according to thepresent invention is a single nucleic acid molecule. In a furtherembodiment, the single nucleic acid molecule is present as a multitudeof the single nucleic acid molecule or as a multitude of the singlenucleic acid molecule species.

It will be acknowledged by the ones skilled in the art that the nucleicacid molecule in accordance with the invention preferably consists ofnucleotides which are covalently linked to each other, preferablythrough phosphodiester links or linkages.

It is within the present invention that the nucleic acid moleculeaccording to the present invention comprises two or more stretches orpart(s) thereof that can, in principle, hybridise with each other. Uponsuch hybridisation a double-stranded structure is formed. It will beacknowledged by the ones skilled in the art that such hybridisation mayor may not occur, particularly under in vitro and/or in vivo conditions.Also, in case of hybridisation, such hybridisation does not necessarilyoccur over the entire length of the two stretches where, at least basedon the rules for base pairing, such hybridisation and thus formation ofa double-stranded structure may, in principle, occur. As preferably usedherein, a double-stranded structure is a part of a nucleic acid moleculeor a structure formed by two or more separate strands or two spatiallyseparated stretches of a single strand of a nucleic acid molecule,whereby at least one, preferably two or more base pairs exist which arebase pairing preferably in accordance with the Watson-Crick base pairingrules. It will also be acknowledged by the one skilled in the art thatother base pairing such as Hoogsten base pairing may exist in or mayform such double-stranded structure. It is also to be acknowledged thatthe feature that two stretches hybridize preferably indicates that suchhybridization is assumed to happen due to base complementarity of thetwo stretches regardless of whether such hybridization actually occursin vivo and/or in vitro. In connection with the present invention suchstretches are the first terminal stretch of nucleotides and the secondstretch of nucleotides which, in an embodiment, may hybridize as definedabove.

In a preferred embodiment the term arrangement as used herein, means theorder or sequence of structural or functional features or elementsdescribed herein in connection with the nucleic acids molecule(s)disclosed herein.

It will be acknowledged by the person skilled in the art that thenucleic acids according to the present invention are capable of bindingto both C5a and C5. This binding characteristic arises from the factthat for the identification of the nucleic acids a moiety of C5a wasused which is present in both C5a and C5. Accordingly, the nucleic acidsaccording to the present invention are suitable for the detection ofeither C5a or C5 or both. Also, it will be acknowledged by the personskilled in the art that the nucleic acid molecule according to thepresent invention is an antagonist to both C5 and C5a. Because of thisthe nucleic acids according to the present invention are suitable forthe treatment and prevention, respectively, of any disease which isassociated with or caused by either C5a or C5 or both. The scientificrational may be taken from the prior art which establishes that C5a andC5, respectively, are involved or associated with a variety of diseasesand conditions, respectively, and which is incorporated herein byreference.

The C5a binding nucleic acid molecule of present invention disclosedherein have been shown to recognize C5a in the context of C5 (seeExample 1). Therefore, it was investigated whether C5 cleavage to theanaphylatoxin C5a and C5b, which is part of the membrane attack complex(MAC) is inhibited by C5a binding nucleic acids. The MAC is the ultimateproduct of the complement cascade: a pore consisting of C5b-9. MAC isbelieved to insert into the cytoplasmic membranes of pathogens and killthem by induction of cytoplasmic leakage. The assay for C5 cleavage wasachieved by using a complement-dependent sheep erythrocyte hemolysistest. The C5a binding molecule of the invention did not inhibithemolysis (see Example 6). The C5a binding nucleic acid molecule of theinvention does not interfere with C5 cleavage and MAC formation and aretherefore selective antagonists of C5a only. If used as a medicament,this may be advantageous in many diseases, since the formation of theMAC that is beneficial in pathogen defense is not compromised in theirpresence.

The nucleic acid molecule according to the present invention shall alsocomprise nucleic acids which are essentially homologous to theparticular sequences disclosed herein. The term substantially homologousshall be understood such as the homology is at least 75%, preferably85%, more preferably 90% and most preferably more that 95%, 96%, 97%,98% or 99%.

The actual percentage of homologous nucleotides present in the nucleicacid according to the present invention will depend on the total numberof nucleotides present in the nucleic acid. The percent modification canbe based upon the total number of nucleotides present in the nucleicacid.

The homology between two nucleic acid molecules can be determined asknown to the person skilled in the art. More specifically, a sequencecomparison algorithm may be used for calculating the percent sequencehomology for the test sequence(s) relative to the reference sequence,based on the designated program parameters. The test sequence ispreferably the sequence or nucleic acid molecule which is said to behomologous or to be tested whether it is homologous, and if so, to whatextent, to a different nucleic acid molecule, whereby such differentnucleic acid molecule is also referred to as the reference sequence. Inan embodiment, the reference sequence is a nucleic acid molecule asdescribed herein, preferably a nucleic acid molecule having a sequenceaccording to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 90, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 57,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 91 and SEQ ID NO: 92. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith & Waterman (Smith & Waterman, 1981) bythe homology alignment algorithm of Needleman & Wunsch (Needleman &Wunsch, 1970) by the search for similarity method of Pearson & Lipman(Pearson & Lipman, 1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by visual inspection.

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al (Altschul etal. 1990 and Altschul et al, 1997). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (hereinafter “NCBI”). The default parametersused in determining sequence identity using the software available fromNCBI, e.g., BLASTN (for nucleotide sequences) and BLASTP (for amino acidsequences) are described in McGinnis et al (McGinnis et al, 2004).

The nucleic acids according to the present invention shall also comprisenucleic acids which have a certain degree of identity relative to thenucleic acids disclosed herein and defined by their nucleotide sequence.More preferably, the instant invention also comprises those nucleic acidmolecules which have an identity of at least 75%, preferably 85%, morepreferably 90% and most preferably more than 95%, 96%, 97%, 98% or 99%relative to the nucleic acids disclosed herein and defined by theirnucleotide sequence or a part thereof.

The term inventive nucleic acid or nucleic acid according to the presentinvention shall also comprise those nucleic acids comprising the nucleicacids sequences disclosed herein or part thereof, preferably to theextent that the nucleic acids or said parts are involved in the bindingto human C5a. Such nucleic acid is, in an embodiment, one of the nucleicacid molecules described herein, or a derivative and/or a metabolitethereof, whereby such derivative and/or metabolite are preferably atruncated nucleic acid compared to the nucleic acid molecules describedherein. Truncation may be related to either or both of the ends of thenucleic acids as disclosed herein. Also, truncation may be related tothe inner sequence of nucleotides of the nucleic acid, i.e. it may berelated to the nucleotide(s) between the 5′ and the 3′ terminalnucleotide, respectively. Moreover, truncation shall comprise thedeletion of as little as a single nucleotide from the sequence of thenucleic acids disclosed herein. Truncation may also be related to morethan one stretch of the inventive nucleic acid(s), whereby the stretchcan be as little as one nucleotide long. The binding of a nucleic acidaccording to the present invention can be determined by the ones skilledin the art using routine experiments or by using or adopting a method asdescribed herein, preferably as described herein in the example part.

The nucleic acid molecule according to the present invention may beeither a D-nucleic acid molecule or an L-nucleic acid molecule.Preferably, the nucleic acid molecule according to the present inventionis an L-nucleic acid molecule. More preferably, the nucleic acidmolecule of the present invention is a Spiegelmer.

It is also within the present invention that, in an embodiment, each andany of the nucleic acid molecules described herein in their entirety interms of their nucleic acid sequence(s) are limited to the particularindicated nucleotide sequence(s). In other words, the terms “comprising”or “comprise(s)” shall be interpreted in such embodiment in the meaningof containing or consisting of.

It is also within the present invention that the nucleic acids accordingto the present invention are part of a longer nucleic acid whereby thislonger nucleic acid comprises several parts whereby at least one suchpart is a nucleic acid according to the present invention, or a partthereof. The other part(s) of these longer nucleic acids can be eitherone or several D-nucleic acid(s) or one or several L-nucleic acid(s).Any combination may be used in connection with the present invention.These other part(s) of the longer nucleic acid either alone or takentogether, either in their entirety or in a particular combination, canexhibit a function which is different from binding, preferably frombinding to C5a. One possible function is to allow interaction with othermolecules, whereby such other molecules preferably are different fromC5a, such as, e.g., for immobilization, cross-linking, detection oramplification. In a further embodiment of the present invention thenucleic acids according to the invention comprise, as individual orcombined moieties, several of the nucleic acids of the presentinvention. Such nucleic acid comprising several of the nucleic acids ofthe present invention is also encompassed by the term longer nucleicacid.

An L-nucleic acid as used herein is a nucleic acid or nucleic acidmolecule consisting of L-nucleotides, preferably consisting completelyof L-nucleotides.

A D-nucleic acid as used herein is nucleic acid or nucleic acid moleculeconsisting of D-nucleotides, preferably consisting completely ofD-nucleotides.

The terms nucleic acid and nucleic acid molecule are used herein in aninterchangeable manner if not explicitly indicated to the contrary.

Also, if not indicated to the contrary, any nucleotide sequence is setforth herein in 5′→3′ direction.

As preferably used herein any position of a nucleotide is determined orreferred to relative to the 5′ end of a sequence, a stretch or asubstretch containing such nucleotide. Accordingly, a second nucleotideis the second nucleotide counted from the 5′ end of the sequence,stretch and substretch, respectively. Also, in accordance therewith, apenultimate nucleotide is the second nucleotide counted from the 3′ endof a sequence, stretch and substretch, respectively.

Irrespective of whether the nucleic acid molecule of the inventionconsists of D-nucleotides, L-nucleotides or a combination of both withthe combination being e.g. a random combination or a defined sequence ofstretches consisting of at least one L-nucleotide and at least oneD-nucleic acid, the nucleic acid may consist of desoxyribonucleotide(s),ribonucleotide(s) or combinations thereof.

It is also within the present invention that the nucleic acid moleculeconsists of both ribonucleotides and 2′ deoxyribonucleotides. The 2′deoxyribonucleotides and ribonucleotides are shown in FIGS. 22 and 23.In order to distinguish between ribonucleotides and2′deoxyribonucleotides in the sequences of the nucleic acid moleculesaccording to the present invention the following reference code is usedherein.

The nucleic acid molecule according to the present invention consists of2′deoxyribonucleotides, wherein

-   -   dG is 2′deoxy-guanosine-5′-monophosphate,    -   dC is 2′ deoxy-cytidine-5′-monophosphate,    -   dA is 2′deoxy-adenosine-5′-monophosphate,    -   dU is 2′deoxy-uridine 5′monophosphate

The nucleic acid molecule according to the present invention consists ofribonucleotides, wherein

-   -   G is guanosine-5′-monophosphate,    -   C is cytidine 5′-monophosphate,    -   A is adenosine-5′-monophosphate,    -   U is uridine-5′monophosphate.

For definition of ribonucleotide sequence motifs, the IUPACabbreviations for ambiguous nucleotides are used:

S strong G or C; W weak A or U; R purine G or A; Y pyrimidine C or U; Kketo G or U; M imino A or C; B not A C or U or G; D not C A or G or U; Hnot G A or C or U; V not U A or C or G; N all A or G or C or U

Designing the nucleic acid molecule of the invention as an L-nucleicacid molecule is advantageous for several reasons. L-nucleic acidmolecules are enantiomers of naturally occurring nucleic acids.D-nucleic acid molecules, however, are not very stable in aqueoussolutions and particularly in biological systems or biological samplesdue to the widespread presence of nucleases. Naturally occurringnucleases, particularly nucleases from animal cells are not capable ofdegrading L-nucleic acids. Because of this, the biological half-life ofan L-nucleic acid molecule is significantly increased in such a system,including the animal and human body. Due to the lacking degradability ofL-nucleic acid molecules no nuclease degradation products are generatedand thus no side effects arising therefrom observed in such a systemincluding the animal and human body. This aspect distinguishes L-nucleicacid molecules from factually all other compounds which are used in thetherapy of diseases and/or disorders involving the presence of C5. AnL-nucleic acid molecule which specifically binds to a target moleculethrough a mechanism different from Watson Crick base pairing, or anaptamer which consists partially or completely of L-nucleotides,particularly with those parts of the aptamer being involved in thebinding of the aptamer to the target molecule, is also called aspiegelmer. Aptamers and spiegelmers as such are known to a personskilled in the art and are, among others, described in ‘The AptamerHandbook’ (eds. Klussmann, 2006).

It is also within the present invention that the nucleic acid moleculeof the invention, regardless whether it is are present as a D-nucleicacid, L-nucleic acid or D,L-nucleic acid or whether it is DNA or RNA,may be present as single stranded or double stranded nucleic acidmolecule. Typically, the nucleic acid molecule is a single strandednucleic acid molecule which exhibits a defined secondary structure dueto its primary sequence and may thus also form a tertiary structure. Thenucleic acid molecule, however, may also be double stranded in themeaning that two strands which are complementary or partiallycomplementary to each other are hybridised to each other.

The nucleic acid molecule of the invention may be modified. Suchmodification may be related to the single nucleotide of the nucleic acidmolecule and is well known in the art. Examples for such modificationare described by, among others, Venkatesan et al. (Venkatesan et al.,2003) and Kusser (Kusser, 2000). Such modification can be a H atom, a Fatom or O—CH₃ group or NH₂-group at the 2′ position of one, several ofall of the individual nucleotides of which the nucleic acid moleculeconsists. Also, the nucleic acid molecule according to the presentinvention can comprise at least one LNA nucleotide. In an embodiment thenucleic acid molecule according to the present invention consists of LNAnucleotides.

In an embodiment, the nucleic acid molecule according to the presentinvention may be a multipartite nucleic acid molecule. A multipartitenucleic acid molecule as used herein is a nucleic acid molecule whichconsists of at least two separate nucleic acid strands. These at leasttwo nucleic acid strands form a functional unit whereby the functionalunit is a ligand to a target molecule and, preferably an antagonist tothe target molecule, in the instant case of C5a. The at least twonucleic acid strands may be derived from any of the nucleic acidmolecule of the invention by either cleaving a nucleic acid molecule ofthe invention to generate at least two strands or by synthesising onenucleic acid molecule corresponding to a first part of the full-lengthnucleic acid molecule of the invention and another nucleic acid moleculecorresponding to another part of the full-length nucleic acid moleculeof the invention. Depending on the number of parts forming thefull-length nucleic acid molecules the corresponding number of partshaving the required nucleotide sequence will be synthesized It is to beacknowledged that both the cleavage approach and the synthesis approachmay be applied to generate a multipartite nucleic acid molecule wherethere are more than two strands as exemplified above. In other words,the at least two separate nucleic acid strands are typically differentfrom two strands being complementary and hybridising to each otheralthough a certain extent of complementarity between said at least twoseparate nucleic acid strands may exist and whereby such complementaritymay result in the hybridisation of said separate strands.

Finally, it is also within the present invention that a fully closed,i.e. circular structure for the nucleic acid molecule according to thepresent invention is realized, i.e. that the nucleic acid moleculeaccording to the present invention are closed in an embodiment,preferably through a covalent linkage, whereby more preferably suchcovalent linkage is made between the 5′ end and the 3′ end of thenucleic acid sequence of the nucleic acid molecule of the invention asdisclosed herein or any derivative thereof.

A possibility to determine the binding constants of the nucleic acidmolecules according to the present invention is the use of the methodsas described in example 3 and 4 which confirms the above finding thatthe nucleic acids according to the present invention exhibit afavourable K_(D) value range. An appropriate measure in order to expressthe intensity of the binding between the individual nucleic acidmolecule and the target which is in the present case C5a is theso-called K_(D) value which as such as well the method for itsdetermination are known to the one skilled in the art.

Preferably, the K_(D) value shown by the nucleic acids according to thepresent invention is below 1 μM. A K_(D) value of about 1 μM is said tobe characteristic for a non-specific binding of a nucleic acid to atarget. As will be acknowledged by the ones skilled in the art, theK_(D) value of a group of compounds such as the nucleic acids accordingto the present invention is within a certain range. The above-mentionedK_(D) of about 1 μM is a preferred upper limit for the K_(D) value. Thelower limit for the K_(D) of target binding nucleic acids can be aslittle as about 10 picomolar or can be higher. It is within the presentinvention that the K_(D) values of individual nucleic acids binding toC5a is preferably within this range. Preferred ranges can be defined bychoosing any first number within this range and any second number withinthis range. Preferred upper K_(D) values are 250 nM and 100 nM,preferred lower K_(D) values are 50 nM, 10 nM, 1 nM, 100 pM and 10 pM.The more preferred upper K_(D) value is 10 nM, the more preferred lowerK_(D) value is 100 pM.

In addition to the binding properties of the nucleic acid moleculesaccording to the present invention, the nucleic acid molecules accordingto the present invention inhibit the function of the respective targetmolecule which is in the present case C5a. The inhibition of thefunction of C5a—for instance the stimulation of the respective receptorsas described previously—is achieved by binding of nucleic acid moleculesaccording to the present invention to C5a and foaming a complex of anucleic acid molecule according to the present invention and C5a. Suchcomplex of a nucleic acid molecule and C5a cannot stimulate thereceptors that normally are stimulated by C5a, i.e. C5a which is notpresent in a complex with a nucleic acid molecule of the invention.Accordingly, the inhibition of receptor function by nucleic acidmolecules according to the present invention is independent from therespective receptor that can be stimulated by C5a but results frompreventing the stimulation of the receptor by C5a by the nucleic acidmolecules according to the present invention.

A possibility to determine the inhibitory constant of the nucleic acidmolecules according to the present invention is the use of the methodsas described in example 5 which confirms the above finding that thenucleic acids according to the present invention exhibit a favourableinhibitory constant which allows the use of said nucleic acids in atherapeutic treatment scheme. An appropriate measure in order to expressthe intensity of the inhibitory effect of the individual nucleic acidmolecule on interaction of the target which is in the present case C5aand the respective receptor, is the so-called half maximal inhibitoryconcentration (abbr. IC₅₀) which as such as well the method for itsdetermination are known to the one skilled in the art.

Preferably, the IC₅₀ value shown by the nucleic acid molecules accordingto the present invention is below 1 μM. An IC₅₀ value of about 1 μM issaid to be characteristic for a non-specific inhibition of targetfunctions by a nucleic acid molecule. As will be acknowledged by theones skilled in the art, the IC₅₀ value of a group of compounds such asthe nucleic acid molecules according to the present invention is withina certain range. The above-mentioned IC₅₀ of about 1 μM is a preferredupper limit for the IC₅₀ value. The lower limit for the IC₅₀ of targetbinding nucleic acid molecules can be as little as about 10 picomolar orcan be higher. It is within the present invention that the IC₅₀ valuesof individual nucleic acids binding to C5a is preferably within thisrange. Preferred ranges can be defined by choosing any first numberwithin this range and any second number within this range. Preferredupper IC₅₀ values are 250 nM and 100 nM, preferred lower IC₅₀ values are50 nM, 10 nM, 1 nM, 100 pM and 10 pM. The more preferred upper IC₅₀value is 5 nM, the more preferred lower IC₅₀ value is 100 pM.

The nucleic acid molecules according to the present invention may haveany length provided that they are still able to bind to the targetmolecule. It will be acknowledged in the art that there are preferredlengths of the nucleic acids according to the present inventions.Typically, the length is between 15 and 120 nucleotides. It will beacknowledged by the ones skilled in the art that any integer between 15and 120 is a possible length for the nucleic acids according to thepresent invention. More preferred ranges for the length of the nucleicacids according to the present invention are lengths of about 20 to 100nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides,about 38 to 44 nucleotides and about 40 nucleotides.

It is within the present invention that the nucleic acid molecule of thepresent invention comprises a moiety which preferably is a highmolecular weight moiety and/or which preferably allows to modify thecharacteristics of the nucleic acid molecule in terms of, among others,residence time in the animal body, preferably the human body. Aparticularly preferred embodiment of such modification is PEGylation andHESylation of the nucleic acids according to the present invention. Asused herein PEG stands for poly(ethylene glycole) and HES forhydroxyethly starch. PEGylation as preferably used herein is themodification of a nucleic acid molecule according to the presentinvention whereby such modification consists of a PEG moiety which isattached to a nucleic acid molecule according to the present invention.HESylation as preferably used herein is the modification of a nucleicacid molecule according to the present invention whereby suchmodification consists of a HES moiety which is attached to a nucleicacid molecule according to the present invention. These modifications aswell as the process of modifying a nucleic acid molecule using suchmodifications, is described in European patent application EP 1 306 382,the disclosure of which is herewith incorporated in its entirety byreference.

In the case of PEG being such high molecular weight moiety the molecularweight is preferably about 20,000 to about 120,000 Da, more preferablyfrom about 30,000 to about 80,000 Da and most preferably about 40,000Da. In the case of HES being such high molecular weight moiety themolecular weight is preferably from about 50 kDa to about 1000 kDa, morepreferably from about 100 kDa to about 700 kDa and most preferably from200 kDa to 500 kDa. HES exhibits a molar substitution of 0.1 to 1.5,more preferably of 1 to 1.5 and exhibits a substitution grade expressedas the C2/C6 ratio of approximately 0.1 to 15, preferably ofapproximately 3 to 10. The process of HES modification is, e.g.,described in German patent application DE 1 2004 006 249.8 thedisclosure of which is herewith incorporated in its entirety byreference.

The modification can, in principle, be made to the nucleic acid moleculeof the present invention at any position thereof. Preferably suchmodification is made either to the 5′-terminal nucleotide, the3′-terminal nucleotide and/or any nucleotide between the 5′ nucleotideand the 3′ nucleotide of the nucleic acid molecule.

The modification and preferably the PEG and/or HES moiety can beattached to the nucleic acid molecule of the present invention eitherdirectly or indirectly, preferably indirectly through a linker. It isalso within the present invention that the nucleic acid moleculeaccording to the present invention comprises one or more modifications,preferably one or more PEG and/or HES moiety. In an embodiment theindividual linker molecule attaches more than one PEG moiety or HESmoiety to a nucleic acid molecule according to the present invention.The linker used in connection with the present invention can itself beeither linear or branched. This kind of linkers are known to the onesskilled in the art and are further described in international patentapplications WO2005/074993 and WO2003/035665.

In a preferred embodiment the linker is a biodegradable linker. Thebiodegradable linker allows to modify the characteristics of the nucleicacid molecule according to the present invention in terms of, amongother, residence time in an animal body, preferably in a human body, dueto release of the modification from the nucleic acid molecule accordingto the present invention. Usage of a biodegradable linker may allow abetter control of the residence time of the nucleic acid moleculeaccording to the present invention. A preferred embodiment of suchbiodegradable linker is a biodegradable linker as described in, but notlimited to, international patent applications WO2006/052790,WO2008/034122, WO2004/092191 and WO2005/099768.

It is within the present invention that the modification or modificationgroup is a biodegradable modification, whereby the biodegradablemodification can be attached to the nucleic acid molecule of the presentinvention either directly or indirectly, preferably through a linker.The biodegradable modification allows modifying the characteristics ofthe nucleic acid molecule according to the present invention in termsof, among other, residence time in an animal body, preferably in a humanbody, due to release or degradation of the modification from the nucleicacid molecule according to the present invention. Usage of abiodegradable modification may allow a better control of the residencetime of the nucleic acid molecule according to the present invention. Apreferred embodiment of such biodegradable modification is biodegradableas described in, but not restricted to, international patentapplications WO2002/065963, WO2003/070823, WO2004/113394 andWO2000/41647, preferably in WO2000/41647, page 18, line 4 to 24.

Beside the modifications as described above, other modifications can beused to modify the characteristics of the nucleic acid moleculeaccording to the present invention, whereby such other modifications maybe selected from the group of proteins, lipids such as cholesterol andsugar chains such as amylase, dextran etc.

Without wishing to be bound by any theory, by modifying the nucleic acidmolecule according to the present invention with a high molecular weightmoiety such as a polymer and more particularly one or several of thepolymers disclosed herein, which are preferably physiologicallyacceptable, the excretion kinetic of the thus modified nucleic acidmolecule of the invention from an animal or human body to which themodified nucleic acid molecule of the invention is administered ischanged is changed. More particularly, due to the increased molecularweight of the tus modified nucleic acid molecule of the invention anddue to the nucleic acid molecule of the invention not being subject tometabolism particularly when in the L form, i.e. being an L-nucleic acidmolecule, excretion from an animal body, preferably from a mammalianbody and more preferably from a human body is decreased. As excretiontypically occurs via the kidneys, the present inventors assume that theglomerular filtration rate of the thus modified nucleic acid molecule issignificantly reduced compared to a nucleic acid molecule not havingthis kind of high molecular weight modification which results in anincrease in the residence time of the modified nucleic acid molecule inthe animal body. In connection therewith it is particularly noteworthythat, despite such high molecular weight modification the specificity ofthe nucleic acid molecule according to the present invention is notaffected in a detrimental manner. Insofar, the nucleic acid moleculeaccording to the present invention has among others, the surprisingcharacteristic—which normally cannot be expected from a pharmaceuticallyactive compound—that a pharmaceutical formulation providing for asustained release is not necessarily required for providing a sustainedrelease of the nucleic acid molecule according to the present invention.Rather, the nucleic acid molecule according to the present invention inits modified form comprising a high molecular weight moiety, can as suchalready be used as a sustained release-formulation as it acts, due toits modification, already as if it was released from a sustained-releaseformulation. Insofar, the modification(s) of the nucleic acid moleculeaccording to the present invention as disclosed herein and the thusmodified nucleic acid molecule according to the present invention andany composition comprising the same may provide for a distinct,preferably controlled pharmacokinetics and biodistribution thereof. Thisalso includes residence time in the circulation of the animal and humanbody and distribution to tissues in such animal and human. Suchmodifications are further described in the patent applicationWO2003/035665.

However, it is also within the present invention that the nucleic acidmolecule according to the present invention does not comprise anymodification and particularly no high molecular weight modification suchas PEG or HES. Such embodiment is particularly preferred when thenucleic acid molecule according to the present invention showspreferential distribution to any target organ or tissue in the body orwhen a fast clearance of the nucleic acid molecule according to thepresent invention from the body after administration is desired. Anucleic acid molecule according to the present invention as disclosedherein with a preferential distribution profile to any target organ ortissue in the body would allow establishment of effective localconcentrations in the target tissue while keeping systemic concentrationof the nucleic acid molecule low. This would allow the use of low doseswhich is not only beneficial from an economic point of view, but alsoreduces unnecessary exposure of other tissues to the nucleic acidmolecule, thus reducing the potential risk of side effects. Fastclearance of the nucleic acid molecule according to the presentinvention from the body after administration might be desired, amongothers, in case of in vivo imaging or specific therapeutic dosingrequirements using the nucleic acid molecule according to the presentinvention or medicaments comprising the same.

The inventive nucleic acids, which are also referred to herein as thenucleic acids according to the present invention, and/or the antagonistsaccording to the present invention may be used for the generation ormanufacture of a medicament. Such medicament or a pharmaceuticalcomposition according to the present invention contains at least one ofthe inventive nucleic acids, optionally together with furtherpharmaceutically active compounds, whereby the inventive nucleic acidpreferably acts as pharmaceutically active compound itself. Suchmedicaments comprise in preferred embodiments at least apharmaceutically acceptable carrier. Such carrier may be, e.g., water,buffer, PBS, glucose solution, preferably a 5% glucose salt balancedsolution, starch, sugar, gelatine or any other acceptable carriersubstance. Such carriers are generally known to the one skilled in theart. It will be acknowledged by the person skilled in the art that anyembodiments, use and aspects of or related to the medicament of thepresent invention is also applicable to the pharmaceutical compositionof the present invention and vice versa.

The indication, diseases and disorders for the treatment and/orprevention of which the nucleic acids, the pharmaceutical compositionsand medicaments in accordance with or prepared in accordance with thepresent invention result from the involvement, either direct orindirect, of C5a in the respective pathogenetic mechanism.

The local release of C5a at sites of inflammation results in powerfulpro-inflammatory stimuli. Thus, neutralization of C5a might bebeneficial in many acute or chronic conditions, such as immune complexassociated diseases in general (Heller et al., 1999); neurodegenerationand inflammation, e.g. in Alzheimer's disease (Bonifati & Kishore,2007), where the complement C5a receptor antagonist PMX205 improvedbehavioral parameters and a reduction of pathological markers such asfibrillar deposits and activated glia (Fonseca et al. 2009). Otherinflammatory diseases with C5a involvement are systemic lupuserythematosus (Jacob et al. 2010a; Jacob et al. 2010b), asthma (Kohl,2001); secondary damages of trauma (Yao et al. 1998); septic shock(Huber-Lang et al., 2001); systemic inflammtory response syndrome(SIRS); multiorgan failure (MOF); acute respiratory distress syndrome(ARDS); inflammatory bowel syndrome (IBD) (Woodruff et al., 2003);immune-complex-mediated renal disease (Wang, 2006), e.g. as acomplication of systemic lupus erythematosus (Manderson et al, 2004);infections and their consequences (e.g. vascular leakage or bone losssuch as bone loss secondary to periodontitis (Breivik et al. 2011));severe burns (Piccolo et al., 1999); reperfusion injury of organs suchas heart, spleen, bladder, pancreas, stomach, lung, liver, kidney,limbs, brain, sceletal muscle or intestine (Riley et al., 2000) (Gueleret al. 2008; Khan et al. 2011; van der Pals et al. 2010; Zheng et al.2008) that may lead amongst others to delayed graft function (Lewis etal, 2008) or fibrosis and/or remodelling of the organ, e.g. after aninfarction of heart, brain or lung leading to secondary damage;psoriasis (Bergh et al., 1993); myocarditis; multiple sclerosis(Muller-Ladner et al., 1996); paroxysmal nocturnal hemoglobinuria (PNH),hemolysis, thromboembolism (Hillmem et al. 2007) and rheumatoidarthritis (RA) (Woodruff et al., 2002), resection of renal cellcarcinoma and activation of osteoclasts promoting bone destruction, e.g.resulting in osteoarthtitis or delayed healing. Complement C5a has alsobeen found in elevated amounts in drusen in age-related maculardegeneration and it has been shown to lead to increased VEGF-expressionand to promote choroidal neovascularization that may lead to visionimpairment and loss (Nozaki et al, 2006).

Activation of the complement system has also been shown to raisesusceptibility to develop cerebral malaria in a mouse model. C5a or C5areceptor blockade using serum from mice immunized with theses moleculesconferred resistance to cerebral malaria. Therefore, blocking the C5 C5aaxis may be beneficial in the prevention of developing malaria,especially cerebral malaria in humans (Patel et al. 2008).

Autoimmune inflammatory diseases with complement involvement have beenreviewed recently (Chen et al. 2010). An expert review on possible andalready pursued complement-targeted therapies appeared in NatureBiotechnology (Ricklin & Lambris, 2007). An update was published inMolecular Medicine in 2011 (Ehrnthaller et al. 2011).

Of course, because the C5a binding nucleic acids according to thepresent invention interact with or bind to human C5a, a skilled personwill generally understand that the C5a binding nucleic acids accordingto the present invention can easily be used for the treatment,prevention and/or diagnosis of any disease of humans and animals asdescribed herein. In connection therewith, it is to be acknowledged thatthe nucleic acid molecules according to the present invention can beused for the treatment and prevention of any of the diseases, disorderor condition described herein, irrespective of the mode of actionunderlying such disease, disorder and condition.

In the following, and without wishing to be bound by any theory, therational for the use of the nucleic acid molecules according to thepresent invention in connection with the various diseases, disorders andconditions is provided, thus rendering the claimed therapeutic,preventive and diagnostic applicability of the nucleic acid moleculesaccording to the present invention plausible. In order to avoid anyunnecessary repetition, it should be acknowledged that due to theinvolvement of the C5a-C5a receptor axis as outlined in connectiontherewith said axis may be addressed by the nucleic acid moleculesaccording to the present invention such that the claimed therapeutic,preventive and diagnostic effect is achieved. It should furthermore beacknowledged that the particularities of the diseases, disorders andconditions, of the patients and any detail of the treatment regimendescribed in connection therewith, may be subject to preferredembodiments of the instant application.

Myeloid-derived suppressor (abbr. MDS) cells were originally observed incancer patient >30 years ago, their role as spoilers of anti-tumorimmunity is only now being appreciated. A heterogeneous population ofnormal myeloid cells trapped in intermediate stages of differentiation,MDS cells accumulate in the blood, lymph nodes and at tumor sites invirtually all cancer patients. In healthy individuals, these cellsdifferentiate into macrophages, dendritic cells and neutrophils, but thetumors secrete a range of factors that disrupt diffentiation of immuneprogenitor cells (Ostrand-Rosenberg, 2008). As shown for isolated MSDcells from the peripheral blood and the spleens of healthy mice, the MDScells express the C5a receptor on their surface to a similar extent—anabundant expression—to that of their mature counterparts granulocytesand monocytes. As well in tumor bearing mice, the MDS cells express theC5a receptor, but the expression level is lower on the surface of tumorassociated MSD cells than on MSD cells in the peripheral blood andspleen. The reason is that the C5a receptor is internalized in tumorassociated cells as shown by Markiewski et al, a C5a receptor antagonistcan block the function of the C5a receptor on the surface of the MSDcells and led to an impaired tumor growth (Markiewski et al. 2008). C5aalso acts as a suppressor of natural killer cell (NK cell) functionsproviding an explanation for negative impact of complement on tumorsurveillance and NK function disorders in patients with certain immunediseases (Li et al. 2012; Min et al. 2012).

Accordingly, disease and/or disorders and/or diseased conditions for thetreatment and/or prevention of which the medicament according to thepresent invention may be used include, but are not limited to tumorassociated diseases and/or disorders and/or diseased conditions.

In a preferred embodiment, tumor or tumour is the name for a swelling orlesion formed by an abnormal growth of cells (termed neoplastic). Atumor can be benign, pre-malignant or malignant tumor. Moreover a tumorcan be a solid tumor, preferably carcininoma, sarcomaa, aoteoma,fibrosarcoma, and chondrosoma

The tumors which can in particular be treated by a medicament or anucleic acid molecule according to the present invention are preferablythose tumors which are selected from the group comprising tumors of theendocrine system, the eye, the gastrointestinal tract, the genitalsystem, the haematopoietic system (including mixed and embryonictumours), the mammary gland, the nervous system, the respiratory system,the skeleton, the skin, the soft tissues, the urinary outflow system

Preferably, these tumors are selected from the group comprising breastcancer, ovary carcinoma, prostate carcinoma, osteosarcoma, glioblastoma,melanoma, small-cell lung carcinoma and colorectal carcinoma.

It is within the present invention that the medicament andpharmaceutical composition, respectively, containing a nucleic acidaccording to the present inventors may be used for the treatment in suchway.

In a further embodiment, the medicament comprises a furtherpharmaceutically active agent for the treatment of tumors. Such furtherpharmaceutically active compounds are, among others but not limitedthereto, those known to antineoplastic-active substances as alkylatingagents, antimetabolites, antiangiogenic agents, mitose inhibitingagents, topoisomerase inhibitors, inhibitors of the cellular signaltransduction, hormones, antibodies, immune conjugates and fusionproteins.

Other pharmaceutically active compounds are, among others but notlimited thereto, those known to Bleomycin, inhibitors ofthymidylatsynthase such as Raltitrexed and Pemetrexed, enzymes such asL-asparaginase, Miltefosin and ANAgrelid, inhibitors of the proteasomesuch as Bortezomib.

Moreover, the medicament according to the present invention may be usedfor the treatment and/or prevention of chronic obstructive pulmonarydisease.

Chronic obstructive pulmonary disease (abbr. COPD) is a lung ailmentthat is characterized by a persistent blockage of airflow from thelungs. It is an under-diagnosed, life-threatening lung disease thatinterferes with normal breathing and is not fully reversible. COPDincludes a few lung diseases: the most common are chronic bronchitis andemphysema. Many people with COPD have both of these diseases. Theemphysema is a damage to the air sacs at the tips of the airways whatmakes it hard for the body to take in the oxygen it needs. Duringchronic bronchitis the airways are irritated, red, and make too muchsticky mucus. The walls of the airways are swollen and partly block theair from passing through.

Neutrophils are attracted towards a C5a gradient, release superoxideradicals to kill pathogens and release beta-glucuronidase to hydrolysecomplex glucuronide conjugates at the site of inflammation. However,these valuable defense mechanisms can be damaging to the body if theneutrophils are recruited to sites of pathogen-free inflammation, e.g.at reperfused sites after infaction, stroke or organ transplantation orin cases of autoimmune disease, Alzheimer's disease and others that arelisted below.

In turn, excessively high C5a concentrations—especially if they are notonly locally elevated as they appear during sepsis—lead to a systemicactivation of the neutrophils (leading to organ damage) with subsequentexhaustion and deactivation by means of reduced C5a receptor expressionon the neutrophils' cell surface. This renders the patient even morevulnerable to the pathogens that persist in his body (Huber-Lang et al.2002).

A C5a-binding nucleic acid that is also inhibitory for the C5a-mediatedeffects on its receptor (CD88) (as shown by chemotaxis assays usingdifferentiated BAF-3 cells) has therefore the potential to block theabove named consequences of C5a signaling and could prove beneficial aspart of a medicament in a number of diseases and conditions whichaberrant C5a signaling is implicated in. One of these conditions ispolymicrobial sepsis. There is a rich body of literature collectingevidence for the detrimental role of C5a signaling in sepsis (Ward2010b). Nucleic acids according to the present inventions were found toimprove survival of mice and organ function parameters in cecal ligationand puncture (CLP) studies. CLP is a well-established rodent model forpolymicrobial sepsis. Recently the role of complement C5 in sepsisinduced by cecal ligation and puncture was investigated in wild-type andC5-deficient mice (Flierl et al. 2008). C5−/− mice had no survivaladvantage compared to WT mice and displayed a 400-fold increase ofblood-borne bacteria when compared to wild-type mice. These effects werelinked to the inability of C5 (−/−) mice to assemble the terminalmembrane attack complex (MAC). The authors conclude that, during sepsis,selective blockade of C5a or its receptor (rather than C5) seems a morepromising strategy, because C5a-blockade still allows for MAC formationwhile the adverse effects of C5a are prevented. In agreement, geneticdeletion of C5a receptors CD88 and C5L2, pharmacological blockade ofCD88 or pharmacological neutralization of C5a has been shown to beprotective in cecal ligation and puncture (CLP)-induced sepsis (Czermaket al. 1999; Rittirsch et al. 2008). A translational in vitro model ofmeningococcal sepsis using human whole blood confirms that selective C5ainhibition (in contrast to blockade of C5 cleavage) prevents potentiallyharmful leukocyte activation without comprising bacterial clearance(Sprong et al. 2003). Inhibition of C5a prevents multiorgan failure inexperimental sepsis by limiting systemic inflammation, coagulation andother pathogenic mechanisms (Huber-Lang et al. 2001; Huber-Lang et al.2002; Laudes et al. 2002; Rittirsch et al. 2008; Ward 2010a). Thenucleic acids according to this invention, though binding to C5 do notblock C5 cleavage to C5a and C5b which is required for MAC formation. Onthe contray, the inventive nucleic acids occupy the protein C5, whichalso increases terminal plasma half-life and selectively block theaction of C5a once this has been liberated, e.g. by the C5 convertase.In a sepsis model, nucleic acids according to this invention have beenshown to limit inflammation, prevent multi organ failure and edemaformation and to improve survival. Sepsis patients often requiremechanical ventilation due to acute lung injury (ALI) and acuterespiratory distress syndrome (ARDS). ALI/ARDS may also develop from adirect infection of the lung by community- or hospital-acquiredinfections in pneumonia. There is abundant evidence for a pathogenicrole of C5a in ALI/ARDS. C5a induced tissue factor expressioncontributes to fibrin deposition within pulmonary alveoli of ALI/ARDSpatients (Kambas et al. 2008). Experimental ALI is attenuated in C5−/−mice and C5a-neutralisation or silencing of C5aR in the lungs suppressesthe inflammatory response and prevents vascular leakage (Bosmann & Ward2012).

Moreover, other disease and/or disorders and/or diseased conditions forthe treatment and/or prevention of which the medicament according to thepresent invention may be used include, but are not limited to areautoimmune diseases such as rheumatoid arthritis (abbr. RA), ankylosingspodylitis (abbr. AS), systemic lupus erythematosus (abbr. SLE),multiple sclerosis (abbr. MS), psoriasis, alopecia areata, warm and coldautoimmune hemolytic anemia (abbr. AIHA), atypical haemolytic uremia,pernicious anemia, acute inflammatory diseases, autoimmune adrenalitis,chronic inflammatory demyelinating polyneuropathy (abbr. CIDP),Churg-Strauss syndrome, Cogan syndrome, CREST syndrome, pemphigusvulgaris and pemphigus foliaceus, bullous pemphigoid, polymyalgiarheumatica, polymyositis, primary biliary cirrhosis, pancreatitis,peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis,Sjörgensen syndrome, scleroderma, celiac disease, stiff-man syndrome,Takayasu arteritis, transient gluten intolerance, autoimmune uveitis,vitiligo, polychondritis, dermatitis herpetiformis (abbr. DH) orDuhring's disease, fibromyalgia, Goodpasture syndrome, Guillain-Barrésyndrome, Hashimoto thyroiditis, autoimmune hepatitis, inflammatorybowel disease (abbr. IBD), Crohn's disease, colitis ulcerosa, myastheniagravis, immune complex disorders, glomerulonephritis, polyarteritisnodosa, anti-phospholipid syndrome, polyglandular autoimmune syndrome,idiopatic pulmonar fibrosis, idiopathic thrombocytopenic purpura (abbr.ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis,sarcoidosis, autoimmune cardiomyopathy, Lambert-Eaton syndrome, lichensclerosis, Lyme disease, Graves disease, Behçet's disease, Meniere'sdisease, reactive arthritis (Reiter's syndrome); infections with virusessuch as HIV, HBV, HCV, CMV or intracellular parasites such asLeishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella,mycobacteria, Listeria, Toxoplasma and Trypanosoma; secondary damages oftrauma; local inflammation, shock, anaphylactic shock, burn, septicshock, haemorrhagic shock, systemic inflammatory response syndrome(abbr. SIRS), multiple organ failure (abbr. MOF), asthma and allergy,vasculitides such as arteritis temporalis, vasculitis, vascular leakage,and atherosclerosis; acute injuries of the central nervous system,myocarditis, dermatomyositis, gingivitis, acute respiratoryinsufficiency, chronic obstructive pulmonary disease, stroke, myocardialinfarction, reperfusion injury, neurocognitive dysfunction, burn,inflammatory diseases of the eye such as uveitis, age-related maculardegeneration (abbr. AMD), diabetic retinopathy (abbr. DR), diabeticmacular edema (abbr. DME), ocular pemphigoid, keratoconjunctivitis,Stevens-Johnson syndrome, and Graves ophthalmopathy; localmanifestations of systemic diseases, inflammatory diseases of thevasculature, acute injuries of the central nervous system, type 1 and 2diabetes, the manifestations of diabetes, SLE, and rheumatic disease inthe eye, brain, vasculature, heart, lung, kidneys, liver,gastrointestinal tract, spleen, skin, bones, lymphatic system, blood orother organ systems, for the prevention and/or support and/orpost-operative treatment of coronary artery bypass graft (abbr. CABG),off-pump coronary artery bypass graft (abbr. OPCABG), minimally invasivedirect coronary artery bypass graft (abbr. MIDCAB), percutaneoustransluminal coronary angioplasty (abbr. PTCA), thrombolysis, organtransplantation, and vessel clamping surgery; for the prevention oforgan damage of a transplanted organ or of an organ to be transplantedor for use of treatment of transplant rejection for transplanted organssuch as liver, kidney, intestine, lung, heart, skin, limb, cornea,Langerhans islet, bone marrow, blood vessels and pancreas; fetalrejection.

The various diseases and disorders for the treatment and/or preventionof which the nucleic acids can be used, may be grouped as follows:

1. Autoimmune/inflammatory diseases

1.1 Systemic autoimmune and/or inflammatory diseases comprising allergy,septic shock, secondary damages of trauma, warm and cold autoimmunehemolytic anemia (abbr. AIHA), systemic inflammatory response syndrome(abbr. SIRS), hemorrhagic shock, diabetes type 1, diabetes type 2, themanifestations of diabetes, diffuse scleroderma, periodontitis and itsassociates bone loss, polychondritis, polyglandular autoimmune syndrome,rheumatoid arthritis, systemic lupus erythematosus (abbr. SLE) andmanifestations thereof, reactive arthritis (also known as Reiter'ssyndrome).

1.2 Autoimmune and/or inflammatory diseases of the gastro-intestinaltract comprising Crohn's disease, colitis ulcerosa, celiac disease,transient gluten intolerance, inflammatory bowel disease (abbr. IBD),pancreatitis, gastrointestinal allergic hypersensitivity, necrotizingenterocolitis.

1.3 Autoimmune and/or inflammatory diseases of the skin comprisingpsoriasis, urticaria, dermatomyositis, pemphigus vulgaris, pemphigusfoliaceus, bullous pemphigoid, morphea/linear scleroderma, vitiligo,dermatitis herpetiformis (abbr. DH) or Duhring's disease, lichensclerosis.

1.4 Autoimmune and/or inflammatory diseases of the vasculaturecomprising vasculitides (preferably arteritis temporalis), vasculitis,Henoch Schönlein purpura, anti-neutrophil cytoplasmic antibody(ANCA)-associated vasculitis, vascular leakage, polymyalgia rheumatica,atherosclerosis, Churg-Strauss syndrome, Takayasu arteritis, Goodpasturesyndrome (=antiglomerular basement membrane disease: mostly affectingthe kidneys glomeruli and the lungs), glomerulonephritis, polyarteritisnodosa, Behçet's disease 1.5 Autoimmune and/or inflammatory diseases ofthe nervous system comprising multiple sclerosis (abbr. MS), chronicinflammatory demyelinating polyneuropathy (abbr. CIDP), neurocognitivedysfunction, stiff-man syndrome, Guillain-Barré syndrome, myastheniagravis, Lambert-Eaton syndrome, neuromyelitis optica (Devic syndrome).

1.6 Muscular skeletal autoimmune and/or inflammatory diseases comprisingrheumatoid arthritis, rheumatic disease in the eye, brain, lung,kidneys, heart, liver, gastrointestinal tract, spleen, skin, bones,lymphatic system, blood or other organs, ankylosing spodylitis (abbr.AS), sarcoidosis, periodontitis and associated bone loss, polymyalgiarheumatica, polymyositis, psoriatic arthritis, rheumatic fever,polychondritis, fibromyalgia, juvenile rheumatoid arthritis, Lymedisease, reactive arthritis (also known as Reiter's syndrome).

1.7 Other autoimmune and/or inflammatory diseases comprise Cogansyndrome, autoimmune adrenalitis, immune complex disordes, Ménière'sdisease, local inflammations, alopecia areata, acute inflammatorydiseases, primary biliary cirrhosis, Sjörgen's syndrome, scleroderma,diffuse scleroderma, CREST syndrome, Morphea/linear scleroderma,autoimmune uveitis, Hashimoto thyroiditis (autoimmune thyroiddestruction), Graves disease, autoimmune hepatitis, non-alcoholicsteatohepatitis, glomerulonephritis, peritonitis, anti-phospholipidsyndrome, idiopathic pulmonary fibrosis, renal fibrosis, hepaticfibrosis, autoimmune infertility, fetal rejection or miscarriage andgraft-versus-host disease.

2. Diseases of the eye comprising uveitis, conjunctivitis, age-relatedmacular degeneration (abbr. AMD), diabetic retinopathy (abbr. DR),diabetic macular edema (abbr. DME), retinal vessel occlusion, glaucoma,cataract, autoimmune retinal and intraocular inflammatory disease,ocular pemphigoid, keratoconjunctivitis, Stevens-Johnson syndrome, andGraves ophthalmopathy.

3. Reperfusion injuries and transplant rejections comprising stroke,myocardial infarction, reperfusion injuries, posttransplant thromboticmicroangiopathy or organ damage to transplanted organs, such as liver(Arumugam et al. 2004), kidney (Arumugam et al. 2003), intestine, lung,heart, skin, limb cornea, islets of Langerhans (Tokodai et al. 2010),bone marrow, blood vessels and pancreas, kidney damage after organ orbone marrow transplantation.

4. Prevention of transplant rejection comprising transplant rejection oftransplanted organs, such as liver, kidney, intestine, lung, heart,skin, limb, cornea, islets of Langerhans, bone marrow, blood vessels andpancreas.

5. Cardiovascular diseases comprising atherosclerosis, myocarditis,myocardial infarction, stroke, pulmonary arterial hypertension (PAH),Abdominal Aortic Aneurism, inflammatory diseases of the vasculature,vasculitides, preferably arteritis temporalis, vasculitis, vascularleakage, the manifestations of diabetes, pre-eclempsia, autoimmunecardiomyopathy, vein host disease, arrythmogenic right ventrivulardysplasia/cardiomyopathy, for the prevention and/or support and/orpost-operative treatment of coronary artery bypass graft (abbr. CABG).

6. Metabolic dysfunction comprising insulin resistance, glucoseintolerance, adipose inflammation, and cardiovascular dysfunction indiet-induced obesity.

7. Respiratory diseases comprising asthma, acute respiratoryinsufficiency, acute lung injury, transfusion related lung injury, adultrespiratory distress syndrome, chronic obstructive pulmonary disease,ventilator-induced lung injury, pneumonia and complications thereof.

8. Inflammatory diseases comprising inflammatory disease of the eye,autoimmune uveitis (Copland et al. 2010), conjunctivitis, vernalconjunctivitis, local manifestations of systemic diseases.

9. Acute reactions comprising secondary damages of trauma and fractures,shock, burn, anaphylactic shock, hemorrhagic shock, multiple organfailure (abbr. MOF), acute injuries of the central nervous system, acuteinjuries of the central nervous system, acute damage due to excessiveC5a production by an activated coagulation system such as after organ orislet transplantation,

10. pain, acute pain, chronic pain, neuropathic pain, morphine toleranceand withdrawal-induced hyperalgesia.

11. Neurological and neurodegenerative disorders comprisingneuropathies, Amyotrophic Lateral Sclerosis (ALS), Alzheimer's diseaseand Parkinson's disease (Farkas et al. 1998).

12. Infectious diseases comprising

12.1 bacterial infections, preferably meningitis, Lyme disease, reactivearthritis (also known as Reiter's syndrome), urinary tract and kidneyinfection, sepsis and its complications such as organ failure, cardiacdysfunction, systemic hypoperfusion, acidosis, adult respiratorydistress syndrome, infections with intracellular pathogens (Klos et al.2009),

12.2 viral infections, preferably HIV, HBV, HCV, CMV, viral meningitis

12.3 intracellular parasites, preferably Leishmania, Rickettsia,Chlamydia, Coxiella, plasmodium, especially cerebral malaria, Brucella,mycobacteria, Listeria, Toxoplasma and Trypanosoma.

13. Hemotological diseases comprising diseases associated withactivation of coagulation and fibrinolytic systems disseminatedintravascular coagulation (DIC) and/or thrombosis, pernicious anemia,warm and cold autoimmune hemolytic anemia (abbr. AIHA),anti-phospholipid syndrome and its associated complications, arterialand venous thrombosis, pregnancy complications such as recurrentmiscarriage and fetal death, preeclampsia, placental insufficiency,fetal growth restriction, cervical remodeling and preterm birth,idiopathic thrombocytopenic purpura (abbr. ITP), atypical hemolyticuremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH) andallergic transfusion reactions.

14. Clinical complications associated with activation by biomaterials ofcomplement and coagulation cascades occurring in procedures comprisinghemodialysis, apheresis, visco-supplementation of arthritic joints,cardiopulmonary bypass, prosthetic vascular grafts and use of cardiovascular devices.

The nucleic acids according to the present invention may also be used inan intra-operative manner to avoid deleterious effects of the patient'simmune system, more preferably for the prevention and/or support and/orpost-operative treatment of coronary artery bypass graft (abbr. CABG),off-pump coronary artery bypass graft (abbr. OPCABG), minimally invasivedirect coronary artery bypass graft (abbr. MIDCAB), percutaneoustransluminal coronary angioplasty (abbr. PTCA), thrombolysis, organtransplantation, brain and spinal cord surgery, reconstructive surgery,and vessel clamping surgery, during any treatment with artificialventilation or ventilation assistance to avoid lung ventilator-inducedlung injury or secondary damages, such as vascular leakage and/oremphysema, for the prevention of organ damage of a transplanted organ orof an organ to be transplanted or for use of treatment of transplantrejection and reperfusion injury for transplanted organs, such as liver,kidney, intestine, lung, heart, skin, limb, cornea, islets ofLangerhans, bone marrow, blood vessels and pancreas.

It is within the present invention that the medicament andpharmaceutical composition, respectively, containing a nucleic acidaccording to the present inventors may be used for the treatment in suchway.

In a further embodiment, the medicament comprises a furtherpharmaceutically active agent. Such further pharmaceutically activecompounds are, among others but not limited thereto, those known tosuppress the immune system such as calcineurin inhibitors, cyclosporinA, methotrexate, azathioprin, tacrolimus, rapamycin, chlorambucil,leflunomide, mycophenolate mofetil, brequinar, mizoribin, thalidomide,or deoxyspergualin. The further pharmaceutically active compound can be,in a further embodiment, also one of those compounds which reducehistamine production such as meclozin, clemastin, dimetinden, bamipin,ketotifen, cetirizin, lovecetirizin, cesloratadin, azelastin,mizolastin, levocabastin, terfenadin, fexofenadin, or ebastin. Suchcompounds can also be, but are not limited to, steroids and arepreferably selected from the group comprising corticosteroids likeprednisone, methylprednisolone, hydrocortisone, dexamethasone,triamcinolone, betamethasone, effervescent, or budesonide. Further, suchcompound can be one or several antibiotics such as, but not restrictedto, aminoglycosides, β-lactam antibiotics, gyrase inhibitors,glycopeptide antibiotics, lincosamide, macrolide antibiotics,nitroimidazole derivatives, polypeptide antibiotics, sulfonamides,trimethoprim and tetracycline. Additionally, more specificanti-inflammatory or antiangiogenic biologics can be used in combinationsuch as bevacizumab, ranibizumab, IL-10, erlizumab, tolermab, rituximab,gomiliximab, basiliximab, daclizumab, HuMax-TAC, visilizumab, HuMaxCD4,clenoliximab, MAX 16H5, TNX 100, toralizumab, alemtuzumab, CY 1788,galiximab, pexelizumab, eculizumab, PMX-53, ETI 104, FG 3019,bertilimumab, 249417 (anti-factor IX) abciximab, YM 337, omalizumab,talizumab, fontolizumab, J695 (anti-IL12), HuMaxIL-15, mepolizumab,elsilimomab, HuDREG, anakinra, Xoma-052, adalimumab, infliximab,certolizumab, afelimomab, CytoFab, AME 527, Vapaliximab, bevacizumab,ranibizumab, vitaxin, belimumab, MLN 1202, volociximab, F200(anti-α5β1), efalizumab, m60.11 (anti.CD11b), etanercept, onercept,rilonacept, abatacept, natalizumab, or siplizumab, tocilizumab,ustekinumab, ABT-874. Finally, the further pharmaceutically active agentmay be a modulator of the activity of any other chemokine which can be achemokine agonist or antagonist or a chemokine receptor agonist orantagonist whereby the chemokine can also be a chemotactic lipid. Anexample is the SIP receptor modulator fingolimod. Alternatively, oradditionally, such further pharmaceutically active agent is a furthernucleic acid according to the present invention. Alternatively, themedicament comprises at least one more nucleic acid which binds to atarget molecule different from C5a or exhibits a function which isdifferent from the one of the nucleic acids according to the presentinvention.

In general the C5a antagonist can be combined with inhibitors of otherproinflammatory molecules or their receptors. Examples forproinflammatory molecules whose action can be attenuated in combinationwith the C5a antagonist are IL-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12,IL-13, IL-15, IL-16, IL-17, IL-18, IL-23, TNF, α4β7, α5β1, BlyS,cadherin, CCR2, CD11a, CD11b, CD125, CD130, CD16, CD18, CD2, CD20,CD22,CD23, CD25, CD28, CD3, CD30, CD4, CD40, CD40L, CD44, CD45R, CD54, CD62E,CD62L, CD68, CD8, CD80, CD86, CD95, CEP, gastrin-R, C1, C1-esterase, C5,factor D, MBL, complement receptor 1, CRTH2-receptor, CTGF, E- andP-selectin, eotaxin, factor IX, FGF-20, Fgl-2, GM-CSF, GP IIb/IIIareceptor, HMG1, ICAM-1, IgE, thymocytes, IFNγ, IFNr, IP-10, MCP-1, M-CSFreceptor, MIF, MMP9, PDGF-D, SDF-1, TGFβ1, tissue factor, tyrosinekinase receptor, VAP-1, VCAM-1, VEGF, VLA1, von Willebrandt factor,sphingosine 1 Phosphate, ceramide-1 phosphate, and inhibitors ofmitogens, e.g. inhibitors of lysophosphatidic acid.

Finally, the further pharmaceutically active agent may be a modulator ofthe activity of any other chemokine which can be a chemokine agonist orantagonist or a chemokine receptor agonist or antagonist. Alternatively,or additionally, such further pharmaceutically active agent is a furthernucleic acid according to the present invention. Alternatively, themedicament comprises at least one more nucleic acid which binds to atarget molecule different from C5a or exhibits a function which isdifferent from the one of the nucleic acids according to the presentinvention.

It is within the present invention that the medicament is alternativelyor additionally used, in principle, for the prevention of any of thediseases disclosed in connection with the use of the medicament for thetreatment of said diseases. Respective markers therefore, i.e. for therespective diseases are known to the ones skilled in the art.Preferably, the respective marker is C5a.

In one embodiment of the medicament of the present invention, suchmedicament is for use in combination with other treatments for any ofthe diseases disclosed herein, particularly those for which themedicament of the present invention is to be used.

“Combination therapy” (or “co-therapy”) includes the administration of amedicament of the invention and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents, i. e. the medicament ofthe present invention and said second agent. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).

“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to asubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, topical routes, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination selected may be administered by injection while the othertherapeutic agents of the combination may be administered topically.

Alternatively, for example, all therapeutic agents may be administeredtopically or all therapeutic agents may be administered by injection.The sequence in which the therapeutic agents are administered is notnarrowly critical unless noted otherwise. “Combination therapy” also canembrace the administration of the therapeutic agents as described abovein further combination with other biologically active ingredients. Wherethe combination therapy further comprises a non-drug treatment, thenon-drug treatment may be conducted at any suitable time so long as abeneficial effect from the co-action of the combination of thetherapeutic agents and non-drug treatment is achieved. For example, inappropriate cases, the beneficial effect is still achieved when thenon-drug treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

As outlined in general terms above, the medicament according to thepresent invention can be administered, in principle, in any form knownto the ones skilled in the art. A preferred route of administration issystemic administration, more preferably by parenteral administration,preferably by injection. Alternatively, the medicament may beadministered locally. Other routes of administration compriseintramuscular, intraperitoneal, and subcutaneous, per orum, intranasal,intratracheal or pulmonary with preference given to the route ofadministration that is the least invasive, while ensuring efficiency.

Parenteral administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, that are well known to theordinary skill in the art.

Furthermore, preferred medicaments of the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, inhalants, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Other preferred topicalpreparations include creams, ointments, lotions, aerosol sprays andgels, wherein the concentration of active ingredient would typicallyrange from 0.01% to 15%, w/w or w/v.

The medicament of the present invention will generally comprise aneffective amount of the active component(s) of the therapy, including,but not limited to, a nucleic acid molecule of the present invention,dissolved or dispersed in a pharmaceutically acceptable medium.Pharmaceutically acceptable media or carriers include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Supplementary active ingredients can also be incorporatedinto the medicament of the present invention.

In a further aspect the present invention is related to a pharmaceuticalcomposition. Such pharmaceutical composition comprises at least one ofthe nucleic acids according to the present invention and preferably apharmaceutically acceptable vehicle. Such vehicle can be any vehicle orany binder used and/or known in the art. More particularly such binderor vehicle is any binder or vehicle as discussed in connection with themanufacture of the medicament disclosed herein. In a further embodiment,the pharmaceutical composition comprises a further pharmaceuticallyactive agent.

The preparation of a medicament and a pharmaceutical composition will beknown to those of skill in the art in light of the present disclosure.Typically, such compositions may be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection; as tablets or other solidsfor oral administration; as time release capsules; or in any other formcurrently used, including eye drops, creams, lotions, salves, inhalantsand the like. The use of sterile formulations, such as saline-basedwashes, by surgeons, physicians or health care workers to treat aparticular area in the operating field may also be particularly useful.Compositions may also be delivered via microdevice, microparticle orsponge.

Upon formulation, a medicament will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

In this context, the quantity of active ingredient and volume ofcomposition to be administered depends on the individual or the subjectto be treated. Specific amounts of active compound required foradministration depend on the judgment of the practitioner and arepeculiar to each individual.

A minimal volume of a medicament required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals.

For instance, for oral administration in the form of a tablet or capsule(e.g., a gelatin capsule), the active drug component, i. e. a nucleicacid molecule of the present invention and/or any furtherpharmaceutically active agent, also referred to herein as therapeuticagent(s) or active compound(s) can be combined with an oral, non-toxic,pharmaceutically acceptable inert carrier such as ethanol, glycerol,water and the like. Moreover, when desired or necessary, suitablebinders, lubricants, disintegrating agents, and coloring agents can alsobe incorporated into the mixture. Suitable binders include starch,magnesium aluminum silicate, starch paste, gelatin, methylcellulose,sodium carboxymethylcellulose and/or polyvinylpyrrolidone, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth or sodium alginate,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol,and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum starches, agar, alginic acid orits sodium salt, or effervescent mixtures, and the like. Diluents,include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, celluloseand/or glycine.

The medicament of the invention can also be administered in such oraldosage forms as timed release and sustained release tablets or capsules,pills, powders, granules, elixirs, tinctures, suspensions, syrups andemulsions. Suppositories are advantageously prepared from fattyemulsions or suspensions.

The pharmaceutical composition or medicament may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating, or coating methods, andtypically contain about 0.1% to 75%, preferably about 1% to 50%, of theactive ingredient.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. The active compound isdissolved in or mixed with a pharmaceutically pure solvent such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form the injectable solution or suspension.Additionally, solid forms suitable for dissolving in liquid prior toinjection can be formulated.

For solid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. Theactive compound defined above, may be also formulated as suppositories,using for example, polyalkylene glycols, for example, propylene glycol,as the carrier. In some embodiments, suppositories are advantageouslyprepared from fatty emulsions or suspensions.

The medicaments and nucleic acid molecules, respectively, of the presentinvention can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, containing cholesterol, stearylamine orphosphatidylcholines. In some embodiments, a film of lipid components ishydrated with an aqueous solution of drug to a form lipid layerencapsulating the drug, what is well known to the ordinary skill in theart. For example, the nucleic acid molecules described herein can beprovided as a complex with a lipophilic compound or non-immunogenic,high molecular weight compound constructed using methods known in theart. Additionally, liposomes may bear such nucleic acid molecules ontheir surface for targeting and carrying cytotoxic agents internally tomediate cell killing. An example of nucleic-acid associated complexes isprovided in U.S. Pat. No. 6,011,020.

The medicaments and nucleic acid molecules, respectively, of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the medicaments andnucleic acid molecules, respectively, of the present invention may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drag, for example, polylactic acid, polyepsiloncapro lactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

If desired, the pharmaceutical composition and medicament, respectively,to be administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and other substances such as for example, sodium acetate, andtriethanolamine oleate.

The dosage regimen utilizing the nucleic acid molecules and medicaments,respectively, of the present invention is selected in accordance with avariety of factors including type, species, age, weight, sex and medicalcondition of the patient; the severity of the condition to be treated;the route of administration; the renal and hepatic function of thepatient; and the particular aptamer or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Effective plasma levels of the nucleic acid according to the presentinvention preferably range from 500 fM to 500 μM in the treatment of anyof the diseases disclosed herein.

The nucleic acid molecules and medicaments, respectively, of the presentinvention may preferably be administered in a single daily dose, everysecond or third day, weekly, every second week, in a single monthly doseor every third month.

It is within the present invention that the medicament as describedherein constitutes the pharmaceutical composition disclosed herein.

In a further aspect the present invention is related to a method for thetreatment of a subject who is in need of such treatment, whereby themethod comprises the administration of a pharmaceutically active amountof at least one of the nucleic acids according to the present invention.In an embodiment, the subject suffers from a disease or is at risk todevelop such disease, whereby the disease is any of those disclosedherein, particularly any of those diseases disclosed in connection withthe use of any of the nucleic acids according to the present inventionfor the manufacture of a medicament.

It is to be understood that the nucleic acid as well as the antagonistsaccording to the present invention can be used not only as a medicamentor for the manufacture of a medicament, but also for cosmetic purposes,particularly with regard to the involvement of C5a in inflamed regionalskin lesions. Therefore, a further condition or disease for thetreatment or prevention of which the nucleic acid, the medicament and/orthe pharmaceutical composition according to the present invention can beused, is inflamed regional skin lesions.

As preferably used herein a diagnostic or diagostic agent or diagnosticmeans is suitable to detect, either directly or indirectly C5a,preferably C5a as described herein and more preferably C5a as describedherein in connection with the various disorders and diseases describedherein. The diagnostic is suitable for the detection and/or follow-up ofany of the disorders and diseases, respectively, described herein. Suchdetection is possible through the binding of the nucleic acids accordingto the present invention to C5a. Such binding can be either directly orindirectly be detected. The respective methods and means are known tothe ones skilled in the art. Among others, the nucleic acids accordingto the present invention may comprise a label which allows the detectionof the nucleic acids according to the present invention, preferably thenucleic acid bound to C5a. Such a label is preferably selected from thegroup comprising radioactive, enzymatic and fluorescent labels. Inprinciple, all known assays developed for antibodies can be adopted forthe nucleic acids according to the present invention whereas thetarget-binding antibody is substituted to a target-binding nucleic acid.In antibody-assays using unlabeled target-binding antibodies thedetection is preferably done by a secondary antibody which is modifiedwith radioactive, enzymatic and fluorescent labels and bind to thetarget-binding antibody at its Fc-fragment. In the case of a nucleicacid, preferably a nucleic acid according to the present invention, thenucleic acid is modified with such a label, whereby preferably such alabel is selected from the group comprising biotin, Cy-3 and Cy-5, andsuch label is detected by an antibody directed against such label, e.g.an anti-biotin antibody, an anti-Cy3 antibody or an anti-Cy5 antibody,or—in the case that the label is biotin—the label is detected bystreptavidin or avidin which naturally bind to biotin. Such antibody,streptavidin or avidin in turn is preferably modified with a respectivelabel, e.g. a radioactive, enzymatic or fluorescent label (like ansecondary antibody).

In a further embodiment the nucleic acid molecules according to theinvention are detected or analysed by a second detection means, whereinthe said detection means is a molecular beacon. The methodology ofmolecular beacon is known to persons skilled in the art. In brief,nucleic acids probes which are also referred to as molecular beacons,are a reverse complement to the nucleic acids sample to be detected andhybridise because of this to a part of the nucleic acid sample to bedetected. Upon binding to the nucleic acid sample the fluorophoricgroups of the molecular beacon are separated which results in a changeof the fluorescence signal, preferably a change in intensity. Thischange correlates with the amount of nucleic acids sample present.

It will be acknowledged that the detection of C5a using the nucleicacids according to the present invention will particularly allow thedetection of C5a as defined herein.

In connection with the detection of C5a a preferred method comprises thefollowing steps:

-   -   (a) providing a sample which is to be tested for the presence of        C5a,    -   (b) providing a nucleic acid according to the present invention,    -   (c) reacting the sample with the nucleic acid, preferably in a        reaction vessel

whereby step (a) can be performed prior to step (b), or step (b) can bepreformed prior to step (a).

In a preferred embodiment a further step d) is provided, which consistsin the detection of the reaction of the sample with the nucleic acid.Preferably, the nucleic acid of step b) is immobilised to a surface. Thesurface may be the surface of a reaction vessel such as a reaction tube,a well of a plate, or the surface of a device contained in such reactionvessel such as, for example, a bead. The immobilisation of the nucleicacid to the surface can be made by any means known to the ones skilledin the art including, but not limited to, non-covalent or covalentlinkages. Preferably, the linkage is established via a covalent chemicalbond between the surface and the nucleic acid. However, it is alsowithin the present invention that the nucleic acid is indirectlyimmobilised to a surface, whereby such indirect immobilisation involvesthe use of a further component or a pair of interaction partners. Suchfurther component is preferably a compound which specifically interactswith the nucleic acid to be immobilised which is also referred to asinteraction partner, and thus mediates the attachment of the nucleicacid to the surface. The interaction partner is preferably selected fromthe group comprising nucleic acids, polypeptides, proteins andantibodies. Preferably, the interaction partner is an antibody, morepreferably a monoclonal antibody. Alternatively, the interaction partneris a nucleic acid, preferably a functional nucleic acid. More preferablysuch functional nucleic acid is selected from the group comprisingaptamers, Spiegelmers, and nucleic acids which are at least partiallycomplementary to the nucleic acid. In a further alternative embodiment,the binding of the nucleic acid to the surface is mediated by amulti-partite interaction partner. Such multi-partite interactionpartner is preferably a pair of interaction partners or an interactionpartner consisting of a first member and a second member, whereby thefirst member is comprised by or attached to the nucleic acid and thesecond member is attached to or comprised by the surface. Themulti-partite interaction partner is preferably selected from the groupof pairs of interaction partners comprising biotin and avidin, biotinand streptavidin, and biotin and neutravidin. Preferably, the firstmember of the pair of interaction partners is biotin.

A preferred result of such method is the formation of an immobilisedcomplex of C5a and the nucleic acid, whereby more preferably saidcomplex is detected. It is within an embodiment that from the complexthe C5a is detected.

A respective detection means which is in compliance with thisrequirement is, for example, any detection means which is specific forthat/those part(s) of the C5a. A particularly preferred detection meansis a detection means which is selected from the group comprising nucleicacids, polypeptides, proteins and antibodies, the generation of which isknown to the ones skilled in the art.

The method for the detection of C5a also comprises that the sample isremoved from the reaction vessel which has preferably been used toperform step c).

The method comprises in a further embodiment also the step ofimmobilising an interaction partner of C5a on a surface, preferably asurface as defined above, whereby the interaction partner is defined asherein and preferably as above in connection with the respective methodand more preferably comprises nucleic acids, polypeptides, proteins andantibodies in their various embodiments. In this embodiment, aparticularly preferred detection means is a nucleic acid according tothe present invention, whereby such nucleic acid may preferably belabelled or non-labelled. In case such nucleic acid is labelled it candirectly or indirectly be detected. Such detection may also involve theuse of a second detection means which is, preferably, also selected fromthe group comprising nucleic acids, polypeptides, proteins andembodiments in the various embodiments described herein. Such detectionmeans are preferably specific for the nucleic acid according to thepresent invention. In a more preferred embodiment, the second detectionmeans is a molecular beacon. Either the nucleic acid or the seconddetection means or both may comprise in a preferred embodiment adetection label. The detection label is preferably selected from thegroup comprising biotin, a bromo-desoxyuridine label, a digoxigeninlabel, a fluorescence label, a UV-label, a radio-label, and a chelatormolecule. Alternatively, the second detection means interacts with thedetection label which is preferably contained by, comprised by orattached to the nucleic acid. Particularly preferred combinations are asfollows:

the detection label is biotin and the second detection means is anantibody directed against biotin, or wherein

the detection label is biotin and the second detection means is anavidin or an avidin carrying molecule, or wherein

the detection label is biotin and the second detection means is astreptavidin or a stretavidin carrying molecule, or wherein

the detection label is biotin and the second detection means is aneutravidin or a neutravidin carrying molecule, or

wherein the detection label is a bromo-desoxyuridine and the seconddetection means is an antibody directed against bromo-desoxyuridine, orwherein

the detection label is a digoxigenin and the second detection means isan antibody directed against digoxigenin, or wherein

the detection label is a chelator and the second detection means is aradio-nuclide, whereby it is preferred that said detection label isattached to the nucleic acid. It is to be acknowledged that this kind ofcombination is also applicable to the embodiment where the nucleic acidis attached to the surface. In such embodiment it is preferred that thedetection label is attached to the interaction partner.

Finally, it is also within the present invention that the seconddetection means is detected using a third detection means, preferablythe third detection means is an enzyme, more preferably showing anenzymatic reaction upon detection of the second detection means, or thethird detection means is a means for detecting radiation, morepreferably radiation emitted by a radio-nuclide. Preferably, the thirddetection means is specifically detecting and/or interacting with thesecond detection means.

Also in the embodiment with an interaction partner of C5a beingimmobilised on a surface and the nucleic acid according to the presentinvention is preferably added to the complex formed between theinteraction partner and the C5a, the sample can be removed from thereaction, more preferably from the reaction vessel where step c) and/ord) are preformed.

In an embodiment the nucleic acid according to the present inventioncomprises a fluorescence moiety and whereby the fluorescence of thefluorescence moiety is different upon complex formation between thenucleic acid and C5a and free C5a.

In a further embodiment the nucleic acid is a derivative of the nucleicacid according to the present invention, whereby the derivative of thenucleic acid comprises at least one fluorescent derivative of adenosinereplacing adenosine. In a preferred embodiment the fluorescentderivative of adenosine is ethenoadenosine.

In a further embodiment the complex consisting of the derivative of thenucleic acid according to the present invention and the C5a is detectedusing fluorescence.

In an embodiment of the method a signal is created in step (c) or step(d) and preferably the signal is correlated with the concentration ofC5a in the sample.

In a preferred aspect, the assays may be performed in 96-well plates,where components are immobilized in the reaction vessels as describedabove and the wells acting as reaction vessels.

The inventive nucleic acid may further be used as starting material fordrug design. Basically there are two possible approaches. One approachis the screening of compound libraries whereas such compound librariesare preferably low molecular weight compound libraries. In anembodiment, the screening is a high throughput screening. Preferably,high throughput screening is the fast, efficient, trial-and-errorevaluation of compounds in a target based assay. In best case theanalysis are carried by a colorimetric measurement. Libraries as used inconnection therewith are known to the one skilled in the art.

Alternatively, the nucleic acid according to the present invention maybe used for rational design of drugs. Preferably, rational drug designis the design of a pharmaceutical lead structure. Starting from the3-dimensional structure of the target which is typically identified bymethods such as X-ray crystallography or nuclear magnetic resonancespectroscopy, computer programs are used to search through databasescontaining structures of many different chemical compounds. Theselection is done by a computer, the identified compounds cansubsequently be tested in the laboratory.

The rational design of drugs may start from any of the nucleic acidaccording to the present invention and involves a structure, preferablya three dimensional structure, which is similar to the structure of theinventive nucleic acids or identical to the binding mediating parts ofthe structure of the inventive nucleic acids. In any case such structurestill shows the same or a similar binding characteristic as theinventive nucleic acids. In either a further step or as an alternativestep in the rational design of drugs the preferably three dimensionalstructure of those parts of the nucleic acids binding to theneurotransmitter are mimicked by chemical groups which are differentfrom nucleotides and nucleic acids. By this mimicry a compound differentfrom the nucleic acids can be designed. Such compound is preferably asmall molecule or a peptide.

In case of screening of compound libraries, such as by using acompetitive assay which are known to the one skilled in the arts,appropriate C5a analogues, C5a agonists or C5a antagonists may be found.Such competitive assays may be set up as follows. The inventive nucleicacid, preferably a Spiegelmer which is a target binding L-nucleic acid,is coupled to a solid phase. In order to identify C5a analogues labelledC5a may be added to the assay. A potential analogue would compete withthe C5a molecules binding to the Spiegelmer which would go along with adecrease in the signal obtained by the respective label. Screening foragonists or antagonists may involve the use of a cell culture assay asknown to the ones skilled in the art.

The kit according to the present invention may comprise at least one orseveral of the inventive nucleic acids. Additionally, the kit maycomprise at least one or several positive or negative controls. Apositive control may, for example, be C5a, particularly the one againstwhich the inventive nucleic acid is selected or to which it binds,preferably, in liquid form. A negative control may, e.g., be a peptidewhich is defined in terms of biophysical properties similar to C5a, butwhich is not recognized by the inventive nucleic acids. Furthermore,said kit may comprise one or several buffers. The various ingredientsmay be contained in the kit in dried or lyophilised form or solved in aliquid. The kit may comprise one or several containers which in turn maycontain one or several ingredients of the kit. In a further embodiment,the kit comprises an instruction or instruction leaflet which providesto the user information on how to use the kit and its variousingredients.

The pharmaceutical and bioanalytical determination of the nucleic acidaccording to the present invention is elementarily for the assessment ofits pharmacokinetic and biodynamic profile in several humours, tissuesand organs of the human and non-human body. For such purpose, any of thedetection methods disclosed herein or known to a person skilled in theart may be used. In a further aspect of the present invention a sandwichhybridisation assay for the detection of the nucleic acid according tothe present invention is provided. Within the detection assay a captureprobe and a detection probe are used. The capture probe is complementaryto the first part and the detection probe to the second part of thenucleic acid according to the present invention. Both, capture anddetection probe, can be formed by DNA nucleotides, modified DNAnucleotides, modified RNA nucleotides, RNA nucleotides, LNA nucleotidesand/or PNA nucleotides.

Hence, the capture probe comprise a sequence stretch complementary tothe 5′-end of the nucleic acid according to the present invention andthe detection probe comprise a sequence stretch complementary to the3′-end of the nucleic acid according to the present invention. In thiscase the capture probe is immobilised to a surface or matrix via its5′-end whereby the capture probe can be immobilised directly at its5′-end or via a linker between of its 5′-end and the surface or matrix.However, in principle the linker can be linked to each nucleotide of thecapture probe. The linker can be formed by hydrophilic linkers ofskilled in the art or by D-DNA nucleotides, modified D-DNA nucleotides,D-RNA nucleotides, modified D-RNA nucleotides, D-LNA nucleotides, PNAnucleotides, L-RNA nucleotides, L-DNA nucleotides, modified L-RNAnucleotides, modified L-DNA nucleotides and/or L-LNA nucleotides.

Alternatively, the capture probe comprises a sequence stretchcomplementary to the 3′-end of the nucleic acid according to the presentinvention and the detection probe comprise a sequence stretchcomplementary to the 5′-end of the nucleic acid according to the presentinvention. In this case the capture probe is immobilised to a surface ormatrix via its 3′-end whereby the capture probe can be immobiliseddirectly at its 3′-end or via a linker between of its 3′-end and thesurface or matrix. However, in principle, the linker can be linked toeach nucleotide of the sequence stretch that is complementary to thenucleic acid according to the present invention. The linker can beformed by hydrophilic linkers of skilled in the art or by D-DNAnucleotides, modified D-DNA nucleotides, D-RNA nucleotides, modifiedD-RNA nucleotides, D-LNA nucleotides, PNA nucleotides, L-RNAnucleotides, L-DNA nucleotides, modified L-RNA nucleotides, modifiedL-DNA nucleotides and/or L-LNA nucleotides.

The number of nucleotides of the capture and detection probe that mayhybridise to the nucleic acid according to the present invention isvariable and can be dependent from the number of nucleotides of thecapture and/or the detection probe and/or the nucleic acid according tothe present invention itself. The total number of nucleotides of thecapture and the detection probe that may hybridise to the nucleic acidaccording to the present invention should be maximal the number ofnucleotides that are comprised by the nucleic acid according to thepresent invention. The minimal number of nucleotides (2 to 10nucleotides) of the detection and capture probe should allowhybridisation to the 5′-end or 3′-end, respectively, of the nucleic acidaccording to the present invention. In order to realize high specificityand selectivity between the nucleic acid according to the presentinvention and other nucleic acids occurring in samples that are analyzedthe total number of nucleotides of the capture and detection probeshould be or maximal the number of nucleotides that are comprised by thenucleic acid according to the present invention.

Moreover the detection probe preferably carries a marker molecule orlabel that can be detected as previously described herein. The label ormarker molecule can in principle be linked to each nucleotide of thedetection probe. Preferably, the label or marker is located at the5′-end or 3′-end of the detection probe, whereby between the nucleotideswithin the detection probe that are complementary to the nucleic acidaccording to the present invention, and the label a linker can beinserted. The linker can be formed by hydrophilic linkers of skilled inthe art or by D-DNA nucleotides, modified D-DNA nucleotides, D-RNAnucleotides, modified D-RNA nucleotides, D-LNA nucleotides, PNAnucleotides, L-RNA nucleotides, L-DNA nucleotides, modified L-RNAnucleotides, modified L-DNA nucleotides and/or L-LNA nucleotides.

The detection of the nucleic acid according to the present invention canbe carried out as follows:

The nucleic acid according to the present invention hybridises with oneof its ends to the capture probe and with the other end to the detectionprobe. Afterwards unbound detection probe is removed by, e. g., one orseveral washing steps. The amount of bound detection probe whichpreferably carries a label or marker molecule, can be measuredsubsequently as, for example, outlined in more detail in WO/2008/052774which is incorporated herein by reference.

As preferably used herein, the term treatment comprises in a preferredembodiment additionally or alternatively prevention and/or follow-up.

As preferably used herein, the terms disease and disorder shall be usedin an interchangeable manner, if not indicated to the contrary.

As used herein, the term comprise is preferably not intended to limitthe subject matter followed or described by such term. However, in analternative embodiment the term comprises shall be understood in themeaning of containing and thus as limiting the subject matter followedor described by such term.

The various SEQ.ID. Nos., the chemical nature of the nucleic acidmolecules according to the present invention and the target moleculesC5a as used herein, the actual sequence thereof and the internalreference number is summarized in the following table.

TABLE 1 SEQ ID Internal NO. Reference Sequence  1 274-B5-002 L-RNAGCCUGAUGUGGUGUUGAAGGGU UGUGGGGUGUCGACGCACAGGC 2 274-D5-002 L-RNAGCCUGAUGUGGUGUUGAGGGGU UGUGGGGUGUCGACGCACAGGC 3 274-C8-002 L-RNAGCCUGAUGUGGUGUUGAAGGGU UGUUGGGUGUCGACGCACAGGC 4 274-C8-002-G14 L-RNAGCCUGAUGUGGUGGUGAAGGGU (= NOX-D19001) UGUUGGGUGUCGACGCACAGGC 5274-C5-002 L-RNA GCCUGAUGUGGUGGUGAGGGGU UGUGGGGUGUCGACGCACAGGC 6274-G6-002 L-RNA GCCUGAUGUGGUGGUGAGGGGA UGUGGGGUGUCGACGCACAGGC 7274-H6-002 L-RNA GCCUGAUGUGGUGUUGAGGGGC UGUGGGGUGUCGACGCACAGGC 8NOX-D19001-D09 L-RNA/L-DNA GCCUGAUG dU GGUGGUGAAGGGUUGUUGGGUGUCGACGCACAGGC 9 NOX-D19001-D16 L-RNA/L-DNA GCCUGAUGUGGUGGU dGAAGGG UUGUUGGGUGUCGACGCACAGGC 10 NOX-D19001-D17 L-RNA/L-DNAGCCUGAUGUGGUGGUG dA AGGG UUGUUGGGUGUCGACGCACAGGC 11 NOX-D19001-D30L-RNA/L-DNA GCCUGAUGUGGUGGUGAAGGGUU GUUGGG dU GUCGACGCACAGGC 12NOX-D19001-D32 L-RNA/L-DNA GCCUGAUGUGGUGGUGAAGGGUU GUUGGGUG dUCGACGCACAGGC 13 NOX-D19001-D40 L-RNA/L-DNA GCCUGAUGUGGUGGUGAAGGGUUGUUGGGUGUCGACGCA dC AGGC 14 NOX-D19001- L-RNA/L-DNA GCCUGAUG dUGGUGGUGAAGGGU D09-30 UGUUGGG dU GUCGACGCACAGGC 15 NOX-D19001-L-RNA/L-DNA GCCUGAUG dU GGUGGUGAAGGGU D09-32 UGUUGGGUG dU CGACGCACAGGC16 NOX-D19001- L-RNA/L-DNA GCCUGAUG dU GGUGGUGAAGGGU D09-40UGUUGGGUGUCGACGCA dC AGGC 17 NOX-D19001- L-RNA/L-DNAGCCUGAUGUGGUGGUGAAGGGUU D30-32 GUUGGG dU G dU CGACGCACAGGC 18NOX-D19001- L-RNA/L-DNA GCCUGAUGUGGUGGUGAAGGGUU D30-40 GUUGGG dUGUCGACGCA dC AGGC 19 NOX-D19001- L-RNA/L-DNA GCCUGAUGUGGUGGUGAAGGGUUD32-40 GUUGGGUG dU CGACGCA dC AGGC 20 NOX-D19001- L-RNA/L-DNA GCCUGAUGdU GGUGGUGAAGGGUU D09-30-32 GUUGGG dU G dU CGACGCACAGGC 21 NOX-D19001-L-RNA/L-DNA GCCUGAUG dU GGUGGUGAAGGGUU D09-30-40 GUUGGG dU GUCGACGCA dCAGGC 22 NOX-D19001- L-RNA/L-DNA GCCUGAUG dU GGUGGUGAAGGGUU D09-32-40GUUGGGUG dU CGACGCA dC AGGC 23 NOX-D19001- L-RNA/L-DNAGCCUGAUGUGGUGGUGAAGGGUUG D30-32-40 UUGGG dU G dU CGACGCA dC AGGC 24NOX-D19001- L-RNA/L-DNA GCCUGAUG dU GGUGGUGAAGGGUU D09-30-32-40 GUUGGGdU G dU CGACGCA dC AGGC 25 NOX-D19001- L-RNA/L-DNA GCCUGAUG dU GGUGGU dGAAGGGU D09-16-30- UGUUGGG dU G dU CGACGCA dC AGGC 32-40 26 NOX-D19001-L-RNA/L-DNA GCCUGAUG dU GGUGGUG dA AGGGUU D09-17-30- GUUGGG dU G dUCGACGCA dC AGGC 32-40 27 NOX-D19001- L-RNA/L-DNA GCCUGAUG dU GGUGGU dGdAAGGGU DO9-16-17- UGUUGGG dU G dU CGACGCA dC AGGC 30-32-40 (= NOX-D19001-6xDNA) 28 NOX-D19001- L-RNA/L-DNA CCUGAUG dU GGUGGU dGdA AGG 6xDNA-007GUUGUUGGG dU G dU CGACGCA d C AGGC 29 NOX-D19001- L-RNA/L-DNA CUGAUG dUGGUGGU dGdA AGGG 6xDNA-008 UUGUUGGG dU G dU CGACGCA dC AGGC 30NOX-D19001- L-RNA/L-DNA UGAUG dU GGUGGU dGdA AGGGUUG 6xDNA-009 UUGGG dUG dU CGACGCA dC AGGC 31 NOX-D19001- L-RNA/L-DNA GAUG dU GGUGGU dGdAAGGGUUG 6xDNA-010 UUGGG dU G dU CGACGCA dC AGGC 32 NOX-D19001-L-RNA/L-DNA GCUGAUG dU GGUGGU dGdA AGGG 6xDNA-011 UUGUUGGG dU G dUCGACGCA dC A GC 33 NOX-D19001- L-RNA/L-DNA GUGAUG dU GGUGGU dGdA AGGGU6xDNA-012 UGUUGGG dU G dU CGACGCA dC AC 34 NOX-D19001- L-RNA/L-DNA UGAUGdU GGUGGU dGdA AGGGUU 6xDNA-013 GUUGGG dU G dU CGACGCA dC A 35NOX-D19001- L-RNA/L-DNA GCCGAUG dU GGUGGU dGdA AGGGU 6xDNA-018 UGUUGGGdU G dU CGACGCA dC GGC 36 NOX-D19001- L-RNA/L-DNA GGCGAUG dU GGUGGU dGdAAGGGU 6XDNA-019 UGUUGGG dU G dU CGACGCA dC GCC 37 NOX-D19001-L-RNA/L-DNA GCGAUG dU GGUGGU dGdA AGGGUU 6xDNA-020 GUUGGG dU G dUCGACGCA dC GC (= NOX-D20001) 38 NOX-D19001- L-RNA/L-DNA CUGAUG dU GGUGGUdGdA AGGGUU 6xDNA-021 GUUGGG dU G dU CGACGCA dC AGC 39 NOX-D19001-L-RNA/L-DNA UGAUG dU GGUGGU dGdA AGGGUUG 6xDNA-022 UUGGG dU G dU CGACGCAdC AGC 40 NOX-D19001- L-RNA/L-DNA CGAUG dU GGUGGU dGdA AGGGUU 6xDNA-023GUUGGG dU G dU CGACGCA dC AGC 41 NOX-D19001- L-RNA/L-DNA GAUG dU GGUGGUdGdA AGGGUUG 6xDNA-024 UUGGG dU G dU CGACGCA dC AGC 42 NOX-D19001-L-RNA/L-DNA GCUGAUG dU GGUGGU dGdA AGGGU 6xDNA-025 UGUUGGG dU G dUCGACGCA dC AC 43 NOX-D19001- L-RNA/L-DNA GCUGAUG dU GGUGGU dGdA AGGGU6xDNA-026 UGUUGGG dU G dU CGACGCA dC C 44 NOX-D19001- L-RNA/L-DNAGCUGAUG dU GGUGGU dGdA AGGGU 6xDNA-027 UGUUGGG dU G dU CGACGCA dCdC A 45NOX-D19001- L-RNA/L-DNA CGAUG dU GGUGGU dGdA AGGGUUG 6xDNA-028 UUGGG dUG dU CGACGCA dC GC 46 NOX-D19001- L-RNA/L-DNA GAUG dU GGUGGU dGdAAGGGUUGU 6xDNA-029 UGGG dU G dU CGACGCA dC GC 47 NOX-D19001- L-RNA/L-DNAGCGAUG dU GGUGGU dGdA AGGGUU 6xDNA-030 GUUGGG dU G dU CGACGCA dC C 48NOX-D19001- L-RNA/L-DNA GCGAUG dU GGUGGU dGdA AGGGUU 6XDNA-032 GUUGGG dUG dU CGACGCA dC 49 NOX-D19001- L-RNA/L-DNA GGAUG dU GGUGGU dGdA AGGGUUG6xDNA-033 UUGGG dU G dU CGACGCA dC C 50 human C5a L-proteinTLQKKIEEIAAKYKHSWKKCCYDGA CWNDETCEQRAARISLGPRCIKAFTECCWASQLRANISHKDMQLGR 51 rat C5a L-protein DLQLLHQKVEEQAAKYKHRVPKKCCYDGARENKYETCEQRVARVTIGPHCIR AFNECCTIADKIRKESHHKGMLLGR 52 mouse C5aL-protein NLHLLRQKIEEQAAKYKHSVPKKCCY DGARVNFYETCEERVARVTIGPLCIRAFNECCTIANKIRKESPHKPVQLGR 53 Human C5, L-proteinTLQKKIEEIAAKYKHSVVKKCCYDG alpha chain ACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLGRL HMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQGI GISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTSG MQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLP LEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGT ISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTH LPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEG MLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSICN SLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGI RKAFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTH PQFRSIVSALKREALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTA YALLTSLNLKDINYVNPVIKWLSEEQRYGGGFYSTQDTINAIEGLTEYSL LVKQLRLSMDIDVSYKHKGALHNYKMTDKNFLGRPVEVLLNDDLIVSTGF GSGLATVHVTTVVHKTSTSEEVCSFYLKIDTQDIEASHYRGYGNSDYKRI VACASYKPSREESSSGSSHAVMDISLPTGISANEEDLKALVEGVDQLFTD YQIKDGHVILQLNSIPSSDFLCVRFRIFELFEVGFLSPATFTVYEYHRPD KQCTMFYSTSNIKIQKVCEGAACKCVEADCGQMQEELDLTISAETRKQTA CKPEIAYAYKVSITSITVENVFVKYKATLLDIYKTGEAVAEKDSEITFIK KVTCTNAELVKGRQYLIMGKEALQIKYNFSFRYIYPLDSLTWIEYWPRDT TCSSCQAFLANLDEFAEDIFLNGC 54 Rhesus monkeyMLQEKIEEIAAKYKHLVVKKCCYDG C5a VRINHDETCEQRAARISVGPRCVKAFTECCVVASQLRANNSHKDLQLGR 55 NOX-D19001- GCGAUGUGGUGGUGAAGGGUUGUU 020GGGUGUCGACGCACGC 56 NOX-D19001- L-RNA/L-DNA GCGAUG dU GGUGGUGAAGGGUUGU1xDNA-020 UGGGUGUCGACGCACGC 57 N0X-D19001- L-RNA/L-DNA GCGAUG dUGGUGGUGAAGGGUUGU 2xDNA-020 UGGG dU GUCGACGCACGC 58 NOX-D19001-L-RNA/L-DNA GCGAUG dU GGUGGUGAAGGGUU 3xDNA-020 GUUGGG dU G dU CGACGCACGC59 NOX-D19001- L-RNA/L-DNA GCGAUG dU GGUGGUGAAGGGUU 2dU-1dC-020 GUUGGGdU GUCGACGCA dC GC (= NOX-D21001) 60 NOX-D19001- L-RNA/L-DNA GCGAUG dUGGUGGUGAAGGGUU 3dU-1dC-020 GUUGGG dU G dU CGACGCA dC GC 61 L-RNA/L-DNAAUGn₁GGUGKUn₂n₃RGGGHU GUKGGGn₄Gn₅CGACGCA wherein n₁ is U or  dU ,n₂ is G or  dG , n₃ is A or  dA , n₄ is U or  dU , n₅ is U or  dU 62L-RNA/L-DNA AUGn₁GGUGUUn₂n₃AGGGUUG UGGGGn₄Gn₅CGACGCA wherein n₁ is U or dU , n₂ is G or  dG , n₃ is A or  dA , n₄ is U or  dU , n₅ is U or  dU63 L-RNA/L-DNA AUGn₁GGUGUUn₂n₃GGGGUUG UGGGGn₄Gn₅CGACGCAwherein n₁ is U or  dU , n₂ is G or  dG , n₃ is A or  dA , n₄ is U or dU , n₅ is U or  dU 64 L-RNA/L-DNA AUGn₁GGUGUUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA wherein n₁ is U or  dU , n₂ is G or  dG , n₃ is A or dA , n₄ is U or  dU , n₅ is U or  dU 65 L-RNA/L-DNAAUGn₁GGUGGUn₂n₃AGGGUUGU UGGGn₄Gn₅CGACGCA wherein n₁ is U or  dU ,n₂ is G or  dG , n₃ is A or  dA , n₄ is U or  dU , n₅ is U or  dU 66L-RNA/L-DNA AUGn₁GGUGGUn₂n₃GGGGUUG UGGGGn₄Gn₅CGACGCA wherein n₁ is U or dU , n₂ is G or  dG , n₃ is A or  dA , n₄ is U or  dU , n₅ is U or  dU67 L-RNA/L-DNA AUGn₁GGUGGUn₂n₃GGGGAUG UGGGGn₄Gn₅CGACGCAwherein n₁ is U or  dU , n₂ is G or  dG , n₃ is A or  dA , n₄ is U or dU , n₅ is U or  dU 68 L-RNA/L-DNA AUGn₁GGUGUUn₂n₃GGGGCUGUGGGGn₄Gn₅CGACGCA wherein n₁ is U or  dU , n₂ is G or  dG , n₃ is A or dA , n₄ is U or  dU , n₅ is U or  dU 69 L-RNA/L-DNAAUGUGGUGKUGARGGGHUGUKG GGUGUCGACGCA 70 L-RNA/L-DNAAUGUGGUGUUGAAGGGUUGUUG GGUGUCGACGCA 71 L-RNA/L-DNAAUGUGGUGGUGAAGGGUUGUUG GGUGUCGACGCA 72 L-RNA/L-DNAAUGUGGUGGUGAGGGGUUGUGG GGUGUCGACGCA 73 L-RNA/L-DNA AUG dUGGUGGUGAAGGGUUGUU GGGUGUCGACGCA 74 L-RNA/L-DNA AUGUGGUGGU dG AAGGGUUGUUGGGUGUCGACGCA 75 L-RNA/L-DNA AUGUGGUGGUG dA AGGGUUGUUG GGUGUCGACGCA 76L-RNA/L-DNA AUGUGGUGGUGAAGGGUUGUUGG G dU GUCGACGCA 77 L-RNA/L-DNAAUGUGGUGGtJGAAGGGUUGUU GGGUG dU CGACGCA 78 L-RNA/L-DNA AUG dUGGUGGUGAAGGGUUGUU GGG dU GUCGACGCA 79 L-RNA/L-DNA AUG dUGGUGGUGAAGGGUUGUU GGGUG dU CGACGCA 80 L-RNA/L-DNA AUGUGGUGGUGAAGGGUUGUUGGG dU G dU CGACGCA 81 L-RNA/L-DNA AUG dU GGUGGUGAAGGGUUGU UGGG dU G dUCGACGCA 82 L-RNA/L-DNA AUG dU GGUGGU dG AAGGGUUGU UGGG dU G dU CGACGCA83 L-RNA/L-DNA AUG dU GGUGGUG dA AGGGUUGU UGGG dU G dU CGACGCA 84L-RNA/L-DNA AUG dU GGUGGU dGdA AGGGUUG UUGGG dU G dU CGACGCA 85274-H6-002 D-RNA GCCUGAUGUGGUGUUGAGGGGC UGUGGGGUGUCGACGCACAGGC 86274-D5-002 D-RNA GCCUGAUGUGGUGUUGAGGGGU UGUGGGGUGUCGACGCACAGGC 87revNOX-D19 L-RNA/L-DNA 40kDaPEG-CGGACACGCAGCUG UGGGUUGUUGGGAAGUGGUGGUGUAGUCCG 88 revNOX-D21 L-RNA/L-DNA 40kDaPEG--CGdCACGCAGCUGdUGGGUUGUUGGGAAGUGGUGGd UGUAGCG 89 Biotinylated D-proteinLLRQKIEEQAAKYKHSVPKKCCY mouse-D- DGARVNFYETCEERVARVTIGPL C5aCIRAFNECCTIANKIRKESPHKP VQLGR- Biotin 90 NOX-D19001- L-RNA/L-DNAC40kDaPEG-GCCUGAUGUGGUGG 5′40kDa- UGAAGGGUUGUUGGGUGUCGACG PEG (= NOX-D19)ACAGGC 91 NOX-D19001- L-RNA/L-DNA 40kDaPEG-GCGAUG dU GGUGG 6xDNA-020- UdGdA AGGGUUGUUGGG dU G dU 5′40kDa-PEG CGACGCA dC GC (= NOX-D20) 92NOX-D19001- L-RNA/L-DNA 40kDaPEG-GCGAUG dU GGUGG 2dU-1dC-UGAAGGGUUGUUGGG dU GUCGA 020-5′40kDa- CGCA dC GC PEG (= NOX-D21) 93human des-ArgC5a L-protein TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKA FTECCVVASQLRANISHKDMQLG 94 mouse des-ArgC5aL-protein NLHLLRQKIEEQAAKYKHSVPKKCCY DGARVNFYETCEERVARVTIGPLCIRAFNECCTIANKIRKESPHKPVQLG 

The present invention is further illustrated by the figures, examplesand the sequence listing from which further features, embodiments andadvantages may be taken, wherein

FIG. 1 shows an alignment of sequences of nucleic acid molecules capableof binding human and mouse C5a including the K_(D) value and relativebinding activity to human and mouse C5a as determined by surface plasmonresonance measurement;

FIG. 2 shows derivatives of nucleic acid molecule NOX-D19001 with asingle ribonucleotide to 2′-deoxyribonucleotide substitution includingthe K_(D) value and relative binding activity to human C5a as determinedby surface plasmon resonance measurement;

FIG. 3 shows derivatives of nucleic acid molecule NOX-D19001 with two,three, four, five or six ribonucleotide to 2′-deoxyribonucleotidesubstitutions including the K_(D) value and relative binding activity tohuman C5a as determined by surface plasmon resonance measurement;

FIGS. 4 A+B show truncations of nucleic acid molecule NOX-D19001-6×DNAincluding the K_(D) value and relative binding activity to human C5a asdetermined by surface plasmon resonance measurement;

FIG. 5 shows derivatives of nucleic acid molecule NOX-D20001 with none,one, two, three or four ribonucleotide to 2′-deoxyribonucleotidesubstitutions including the K_(D) value and relative binding activity tohuman C5a as determined by surface plasmon resonance measurement;

FIG. 6 shows the kinetic evaluation by Biacore measurement of nucleicacid molecules NOX-D19001, NOX-D19001-D09 andNOX-D19001-D09-16-17-30-32-40 (also referred to as NOX-D19001-6×DNA) tohuman C5a whereby the data for 500 nM of Spiegelmer NOX-D19001,NOX-D19001-D09 (abbr. D09) and NOX-D19001-D09-16-17-30-32-40 (abbr.D09-16-17-30-32-40) are shown;

FIG. 7 is a diagram showing the efficacy of 5′-terminal 40 kDa PEGylatedC5a binding Spiegelmers NOX-D19001-5′PEG (also referred as NOX-D19)NOX-D20 (also referred to as NOX-D19001-6×DNA-020-5′40 kDa PEG) inchemotaxis assays, wherein cells were allowed to migrate towards 0.1 nMhuC5a preincubated at 37° C. with various amounts of Spiegelmers,

FIG. 8 shows the kinetic evaluation by Biacore measurement of nucleicacid molecules NOX-D20 (also referred to as NOX-D19001-6×DNA-020-5′40kDa PEG) to human C5a, rat C5a, mouse C5a, monkey C5a; whereby the datafor 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, and 1.95-0 nM ofSpiegelmer NOX-D20 are shown;

FIG. 9 shows the kinetic evaluation by Biacore measurement of nucleicacid molecules NOX-D20 (also referred to as NOX-D19001-6×DNA-020-5′40kDa PEG) to human C5, and human desArg-C5a; whereby the data for 1000,500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, and 1.95-0 nM of SpiegelmerNOX-D20 are shown;

FIG. 10 shows the kinetic evaluation by Biacore measurement of nucleicacid molecules NOX-D21 (also referred to as NOX-D19001-2dU-1dC-020-5′40kDa PEG) to human C5a, human C5, human desArg-C5a, mouse C5a and mousedesArg-C5a; whereby the data for 1000, 500, 250, 125, 62.5, 31.3, 15.6,7.8, 3.9, and 1.95-0 nM of Spiegelmer NOX-D21 binding to human C5a andhuman C5 are shown;

FIG. 11 shows the polypeptide sequence aligment of C5a from human,rhesus monkey, mouse and rat;

FIG. 12A is a diagram showing the efficacy of C5a binding SpiegelmersNOX-D20 in chemotaxis assays with human C5a and mouse C5a, cells wereallowed to migrate towards 0.1 nM huC5a or 0.3 nM mC5a preincubated at37° C. with various amounts of Spiegelmer; wherein a) the cells countswere normalized to the largest value of each data set and depicted aspercent count against Spiegelmer concentration, b) the Spiegelmerconcentrations at which the chemotaxis is inhibited by 50% (IC₅₀) werecalculated using nonlinear regression (four parameter fit) with Prism5software;

FIG. 12B is a diagram showing the efficacy of C5a binding SpiegelmersNOX-D21 in chemotaxis assays with human C5a, cells were allowed tomigrate towards 0.1 nM huC5a preincubated at 37° C. with various amountsof Spiegelmer; wherein a) the cells counts were normalized to thelargest value of each data set and depicted as percent count againstSpiegelmer concentration, b) the Spiegelmer concentrations at which thechemotaxis is inhibited by 50% (IC₅₀) were calculated using nonlinearregression (four parameter fit) with Prism5 software:

FIGS. 13 A&B are diagrams showing the efficacy of C5a bindingSpiegelmers NOX-D19 and NOX-D20 in chemotaxis assays (FIG. 13A), andelastase release assays (FIG. 13B) of primary human PMNs with human C5a;wherein cells were allowed to migrate towards 1 nM huC5a and elastaserelease was stimulated by 30 nM huC5a preincubated at 37° C. withvarious amounts of Spiegelmer;

FIGS. 14 A&B are diagrams showing evaluation of C5 cleavage inhibitionusing a sheep erythrocyte hemolysis assay with Spiegelmers NOX-19 andNOX-D20 (FIG. 14A), and NOX-D21 (FIG. 14B). A positive (C5C6) andnegative controls (revNOX-D19 and revNOX-D21) are shown;

FIG. 15 is a diagram showing survival in the cecal ligation and puncture(CLP) mouse model of polymicrobial sepsis; NOX-D19 at the indicateddoses or vehicle was injected intraperitoneally daily starting rightafter CLP surgery. Sham animals received surgery without CLP, followedby vehicle injections;

FIGS. 16 A&B are diagrams showing serum creatinine levels (FIG. 16A) andblood urea nitrogen (BUN) levels (FIG. 16B) pre-surgery (day −4) and 1day after CLP surgery in mice treated with NOX-D19 at indicated doses,in vehicle treated mice and in sham animals. Serum creatinine and BUNare biomarkers for renal function;

FIGS. 17 A&B are diagrams showing serum levels of alanineaminotransferase (ALT) (FIG. 17A) and serum levels of aspartateaminotransferase (AST) (FIG. 17B) pre-surgery (day −4) and 1 day afterCLP surgery in mice treated with NOX-D19 at indicated doses, in vehicletreated mice and in sham animals. Serum ALT is a biomarker forhepatocellular damage. Serum AST is a biomarker for multiorgan failure;

FIG. 18 is a diagram showing survival in the cecal ligation and puncture(CLP) mouse model of polymicrobial sepsis; NOX-D20 at the indicateddoses or vehicle was injected intraperitoneally daily starting rightafter CLP surgery. One group received a single dose of 1 mg/kg NOX-D20right after CLP surgery followed by daily vehicle injections. Shamanimals received surgery without CLP, followed by vehicle injections.

FIGS. 19A-C are diagrams showing serum creatinine levels (FIG. 19A),blood urea nitrogen (BUN) (FIG. 19B) and serum levels of alanineaminotransferase (ALT) (FIG. 19C) at day 1 after CLP surgery in micetreated with NOX-D20 at indicated doses, in vehicle treated mice and insham animals. Serum creatinine and BUN are biomarkers for renalfunction. Serum ALT is a biomarker for hepatocellular damage;

FIGS. 20A-C are diagrams showing the effect of NOX-D20 treatment atindicated doses on serum lactate dehydrogenase (LDH) (FIG. 20A), abiomarker for tissue injury, vascular leakage (FIG. 20B), and PMNinfiltration into the peritoneum (FIG. 20C) at day 1 after CLP surgery.Vehicle treated mice and in sham animals are shown as controls;

FIG. 21 is a diagram showing survival in a model of ischemia/reperfusioninjury induced acute kidney injury; NOX-D21 at the indicated doses orvehicle was injected intravenously 1 h prior to surgery and subsequentlyintraperitoneally every 24 h for 3 days;

FIG. 22 shows the 2′deoxyribonucleotides that the nucleic acid moleculesaccording to the present invention consist of;

FIG. 23 shows the ribonucleotides that the nucleic acid moleculesaccording to the present invention consist of;

EXAMPLE 1: NUCLEIC ACID MOLECULES CAPABLE OF BINDING HUMAN AND MOUSE C5A

Several C5a binding nucleic acid molecules and derivatives thereof wereidentified: the nucleotide sequences of which are depicted in FIGS. 1 to5. The C5a binding nucleic acid molecules were characterized as

-   -   a) aptamers, i. e. D-nucleic acid molecules using a direct        pull-down assay (Example 3) and/or a comparative competition        pull-down assay (Example 3)    -   b) Spiegelmers, i. e. L-nucleic acid molecules by surface        plasmon resonance measurement (Example 4), and by an in vitro        assay with cells expressing the human C5a receptor (Example 5).        Moreover Spiegelmers were tested for the inhibition of        C5a-induced activation of primary human neutrophils (Example 6)        and in vivo (Example 8, 9 and 10 The Spiegelmers and aptamers        were synthesized as described in Example 2.

The nucleic acid molecules thus generated exhibit slightly differentsequences, whereby the sequences can be summarized or grouped as asequence family.

For definition of ribonucleotide sequence motifs, the IUPACabbreviations for ambiguous nucleotides are used:

S strong G or C; W weak A or U; R purine G or A; Y pyrimidine C or U; Kketo G or U; M imino A or C; B not A C or U or G; D not C A or G or U; Hnot G A or C or U; V not U A or C or G; N all A or G or C or U

If not indicated to the contrary, any nucleic acid sequence or sequenceof stretches, respectively, is indicated in the 5′→3′ direction.

For differentiation between the 2′-deoxyribonucleotides and theribonucleotides the following abbreviations are used:

For 2′-deoxyribonucleotides: dG, dC, dT, dA and dU (see FIG. 22).

For ribonucleotides: G, C, T, U (see FIG. 23).

As depicted in FIG. 1 to FIG. 5 C5a binding nucleic acid moleculescomprise one central stretch of nucleotides defining a potential C5abinding motif, whereby FIG. 1 shows the different sequences of thesequence family, the FIGS. 2 to 5 show derivatives of the nucleic acidmolecule NOX-D19001 including NOX-D20001 (also referred to asNOX-D19001-6×-DNA-020, FIG. 4A) and NOX-D21001 (also referred to asNOX-D19001-2dU-1dC-020, FIG. 5).

In general, C5a binding nucleic acid molecules comprise at the 5′-endand the 3′-end terminal stretches of nucleotides: the first terminalstretch of nucleotides and the second terminal stretch of nucleotides.The first terminal stretch of nucleotides and the second terminalstretch of nucleotides can hybridize to each other, whereby uponhybridization a double-stranded structure is formed. However, suchhybridization is not necessarily given in the molecule in vivo and invitro.

The three stretches of nucleotides of C5a binding nucleic acidmolecules—the first terminal stretch of nucleotides, the central stretchof nucleotides and the second terminal stretch of nucleotides—arearranged to each other in 5′→3′-direction: the first terminal stretch ofnucleotides—the central stretch of nucleotides—the second terminalstretch of nucleotides. However, alternatively, the first terminalstretch of nucleotides, the central stretch of nucleotides and thesecond terminal stretch of nucleotides are arranged to each other in5′→3′-direction: the second terminal stretch of nucleotides—the centralstretch of nucleotides—the first terminal stretch of nucleotides.

The sequences of the defined stretches may be different between the C5abinding nucleic acid molecules which influences the binding affinity toC5a. Based on binding analysis of the different C5a binding nucleic acidmolecules the central stretch of nucleotides and their nucleotidesequences as described in the following are individually and morepreferably in their entirety essential for binding to human C5a.

The C5a binding nucleic acid molecules according to the presentinvention as shown in FIG. 1 consist of ribonucleotides and are shown inFIGS. 1 to 5. The C5a binding nucleic acid molecule 274-H6-002 wastested as aptamer in a comparative competition pull-down assays (forprotocol see example 3) vs. C5a binding nucleic acid 274-D5-002. C5abinding nucleic acid molecule 274-H6-002 showed weaker binding affinityin comparison to C5a binding nucleic acid molecule 274-D5-002. The C5abinding nucleic acid molecules 274-B5-002, 274-D5-002, 274-C8-002,274-C8-002-G14 (=NOX-D19001), 274-05-002 and 274-G6-002 were tested asSpiegelmers for their ability to bind human and mouse C5a by plasmonresonance measurement (see Example 4, FIG. 1).

C5a binding nucleic acid molecule 274-C8-002-G14 (=NOX-D19001) shows thebest binding affinity with a K_(D) of 0.3 nM for mouse C5a and with aK_(D) of 1.38 nM for human C5a (FIG. 1).

The C5a binding nucleic acid molecules 274-B5-002, 274-D5-002,274-C8-002, 274-C8-002-G14 (=NOX-D19001), 274-05-002, 274-G6-002 and274-H6-002 share the sequence

[SEQ ID NO: 69] 5′ AUGUGGUGKUGARGGGHUGUKGGGUGUCGACGCA 3′,

wherein G, A, U, C, H, K, and R are ribonucleotides.

The C5a binding nucleic acid molecules 274-C8-002, 274-C8-002-G14(=NOX-D19001) and 274-05-002 showed the best binding affinity to C5a andcomprise the following sequences for the central stretch:

a) 274-C8-002: [SEQ ID NO: 70] 5′ AUGUGGUGUUGAAGGGUUGUUGGGUGUCGACGCA 3′,b) 274-C8-002-G14: [SEQ ID NO: 71]5′ AUGUGGUGGUGAAGGGUUGUUGGGUGUCGACGCA 3′, c) 274-05-002: [SEQ ID NO: 72]5′ AUGUGGUGGUGAGGGGUUGUGGGGUGUCGACGCA 3′,

wherein d G, A, U and C are ribonucleotides.

The inventors surprisingly showed that the binding affinity of C5abinding nucleic acid molecule NOX-D19001 was improved by replacingribonucleotides by 2′-deoxyribonucleotides within the sequence of thecentral stretch of nucleotides and the second terminal stretch ofnucleotides. In particular replacing up to six ribonucleotides by2′-deoxyribonucleotides in the C5a binding nucleic acid moleculeNOX-D19001 resulted in improved binding affinity to human C5a by afactor of 3.5. In more detail, the inventors have surprisingly foundthat

-   -   a) replacing one ribonucleotide by one 2′-deoxyribonucleotide at        position 4, 11, 12, 25 or 27 in the central stretch of        nucleotides of C5a binding nucleic acid molecule NOX-D19001        resulted in improved binding affinity to human C5a in comparison        to the binding affinity of C5a binding nucleic acid molecule        NOX-D19001 (see FIG. 2; Spiegelmers NOX-D19001-D09,        NOX-D19001-D16, NOX-D19001-D17, NOX-D19001-D30, NOX-D19001-D32);    -   b) replacing one ribonucleotide by one 2′-deoxyribonucleotide at        position 1 in the second terminal stretch of nucleotides of C5a        binding nucleic acid molecule NOX-D19001 resulted in improved        binding affinity to human C5a in comparison to the binding        affinity of C5a binding nucleic acid molecule NOX-D19001 (see        FIG. 2; Spiegelmers NOX-D19001-D40);    -   c) replacing two ribonucleotides by two 2′-deoxyribonucleotide        at position 4/25, 4/27, or 25/27 in the central stretch of        nucleotides of C5a binding nucleic acid molecule NOX-D19001        resulted in improved binding affinity to biotinylated C5a in        comparison to the binding affinity of C5a binding nucleic acid        molecule NOX-D19001 (see FIG. 3; Spiegelmers NOX-D19001-D09-30,        NOX-D19001-D09-32, NOX-D19001-D30-32);    -   d) replacing two ribonucleotides, wherein one ribonucleotide was        replaced by one 2′-deoxyribonucleotide at position 1 in the        second terminal stretch of nucleotides of C5a binding nucleic        acid molecule NOX-D19001 and one ribonucleotide was replaced by        one 2′-deoxyribonucleotide at position 4, 25 or 27 in the        central stretch of nucleotides of C5a binding nucleic acid        molecule NOX-D19001, resulted in improved binding affinity to        human C5a in comparison to the binding affinity of C5a binding        nucleic acid molecule NOX-D19001 (see FIG. 3; Spiegelmers        NOX-D19001-D09-40, NOX-D19001-D30-40, NOX-D19001-D32-40);    -   e) replacing three ribonucleotides, wherein one ribonucleotide        was replaced by one 2′-deoxyribonucleotide at position 1 in the        second terminal stretch of nucleotides of C5a binding nucleic        acid molecule NOX-D19001 and two ribonucleotides were replaced        by two 2′-deoxyribonucleotides at position 4/25, 4/27, 25/27 in        the central stretch of nucleotides of C5a binding nucleic acid        molecule NOX-D19001, resulted in improved binding affinity to        human C5a in comparison to the binding affinity of C5a binding        nucleic acid molecule NOX-D19001 (see FIG. 3; Spiegelmers,        NOX-D19001-D09-30-40, NOX-D19001-D09-32-40,        NOX-D19001-D30-32-40);    -   f) replacing three ribonucleotides by three        2′-deoxyribonucleotide at position 04/25/27 in the central        stretch of nucleotides of C5a binding nucleic acid molecule        NOX-D19001 resulted in improved binding affinity to biotinylated        C5a in comparison to the binding affinity of C5a binding nucleic        acid molecule NOX-D19001 (see FIG. 3; Spiegelmer        NOX-D19001-D09-30-32);    -   g) replacing four ribonucleotides, wherein one ribonucleotide        was replaced by one 2′-deoxyribonucleotide at position 1 in the        second terminal stretch of nucleotides of C5a binding nucleic        acid molecule NOX-D19001 and three ribonucleotides were replaced        by three 2′-deoxyribonucleotides at position 04/25/27 in the        central stretch of nucleotides of C5a binding nucleic acid        molecule NOX-D19001, resulted in improved binding affinity to        human C5a in comparison to the binding affinity of C5a binding        nucleic acid molecule NOX-D19001 (see FIG. 3; Spiegelmer        NOX-D19001-D09-30-32-40);    -   h) replacing five ribonucleotides, wherein one ribonucleotide        was replaced by one 2′-deoxyribonucleotide at position 1 in the        second terminal stretch of nucleotides of C5a binding nucleic        acid molecule NOX-D19001 and four ribonucleotides were replaced        by four 2′-deoxyribonucleotides at position 04/11/25/27 or        04/12/25/27 in the central stretch of nucleotides of C5a binding        nucleic acid molecule NOX-D19001, resulted in improved binding        affinity to human C5a in comparison to the binding affinity of        C5a binding nucleic acid molecule NOX-D19001 (see FIG. 3;        Spiegelmer NOX-D19001-D09-16-30-32-40,        NOX-D19001-D09-17-30-32-40);    -   i) replacing six ribonucleotides, wherein one ribonucleotide was        replaced by one 2′-deoxyribonucleotide at position 1 in the        second terminal stretch of nucleotides of C5a binding nucleic        acid molecule NOX-D19001 and five ribonucleotides were replaced        by five 2′-deoxyribonucleotides at position 04/11/12/25/27 in        the central stretch of nucleotides of C5a binding nucleic acid        molecule NOX-D19001, resulted in improved binding affinity to        human C5a in comparison to the binding affinity of C5a binding        nucleic acid molecule NOX-D19001 (see FIG. 3; Spiegelmer        NOX-D19001-D09-16-17-30-32-40=NOX-D19-001-6×DNA).

Based on the data shown that replacing ribonucleotides by2′-deoxyribonucleotides at several positions of the central stretch ofnucleotides of C5a binding nucleic acid molecules lead to improvedbinding to C5a the central stretch of all tested C5a binding nucleicacid molecules can be summarized in a the following formula

[SEQ ID NO: 61] 5′ AUGn₁GGUGKUn₂n₃RGGGHUGUKGGGn₄Gn₅CGACGCA 3′,

wherein n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅is U or dU

and G, A, U, C, H, K,

and R are ribonucleotides, and dU, dG and dA are2′-deoxyribonucleotides,

wherein

-   -   a) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 62] 5′ AUGn₁GGUGUUn₂n₃AGGGUUGUGGGGn₄Gn₅CGACGCA 3′(see 274-B5-002);

-   -    or    -   b) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 63] 5′ AUGn₁GGUGUUn₂n₃GGGGUUGUGGGGn₄Gn₅CGACGCA 3′(see 274-D5-002);

-   -    or    -   c) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 64] 5′ AUGn₁GGUGUUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA 3′(see 274-C8-002);

-   -    or    -   d) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 65] 5′ AUGn₁GGUGGUn₂n₃AGGGUUGUUGGGn₄Gn₅CGACGCA 3′(see NOX-D19001);

-   -    or    -   e) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 66] 5′ AUGn₁GGUGGUn₂n₃GGGGUUGUGGGGn₄Gn₅CGACGCA 3′(see 274-05-002);

-   -    or    -   f) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 67] 5′ AUGn₁GGUGGUn₂n₃GGGGAUGUGGGGn₄Gn₅CGACGCA 3′(see 274-G6-002);

-   -    or    -   g) in a preferred embodiment the central stretch of nucleotides        comprise the sequence

[SEQ ID NO: 68] 5′ AUGn₁GGUGUUn₂n₃GGGGCUGUGGGGn₄Gn₅CGACGCA 3′(see 274-H6-002).

The C5a binding nucleic acid molecules NOX-D19001-D09, NOX-D19001-D16,NOX-D19001-D17, NOX-D19001-D30, NOX-D19001-D32, NOX-D19001-D09-30,NOX-D19001-D09-32, NOX-D19001-D09-40, NOX-D19001-D30-32,NOX-D19001-D30-40, NOX-D19001-D32-40, NOX-D19001-D09-30-32,NOX-D19001-D09-30-40, NOX-D19001-D09-32-40, NOX-D19001-D30-32-40,NOX-D19001-D09-30-32-40, NOX-D19001-D09-16-30-32-40,NOX-D19001-D09-17-30-32-40, NOX-D19001-D09-16-17-30-32-40 (see FIGS. 2and 3) showed the best binding affinity to C5a and comprise thefollowing sequence for the central stretch of nucleotides:

a) [SEQ ID NO: 73] 5′ AUGdUGGUGGUGAAGGGUUGUUGGGUGUCGACGCA 3′(see NOX-D19001-D09, NOX-D19001-D09-40); or b) [SEQ ID NO: 74]5′ AUGUGGUGGUdGAAGGGUUGUUGGGUGUCGACGCA 3′ (see NOX-D19001-D16); or c)[SEQ ID NO: 75] 5′ AUGUGGUGGUGdAAGGGUUGUUGGGUGUCGACGCA 3′(see NOX-D19001-D17); or d) [SEQ ID NO: 76]5′ AUGUGGUGGUGAAGGGUUGUUGGGdUGUCGACGCA 3′(see NOX-D19001-D30, NOX-D19001-D30-40); or e) [SEQ ID NO: 77]5′ AUGUGGUGGUGAAGGGUUGUUGGGUGdUCGACGCA 3′(see NOX-D19001-D32, NOX-D19001-D32-40); or f) [SEQ ID NO: 78]5′ AUGdUGGUGGUGAAGGGUUGUUGGGdUGUCGACGCA 3′(see NOX-D19001-D09-30, NOX-D19001-D09-30-40); or g) [SEQ ID NO: 79]5′ AUGdUGGUGGUGAAGGGUUGUUGGGUGdUCGACGCA 3′(see NOX-D19001-D09-32, NOX-D19001-D09-32-40); h) [SEQ ID NO: 80]5′ AUGUGGUGGUGAAGGGUUGUUGGGdUGdUCGACGCA 3′(see NOX-D19001-D30-32, NOX-D19001-D30-32-40); or i) [SEQ ID NO: 81]5′ AUGdUGGUGGUGAAGGGUUGUUGGGdUGdUCGACGCA 3′(NOX-D19001-D09-30-32, NOX-D19001-D09-30-32-40); or j) [SEQ ID NO: 82]5′ AUGdUGGUGGUdGAAGGGUUGUUGGGdUGdUCGACGCA 3′(see NOX-D19001-D09-16-30-32-40); or k) [SEQ ID NO: 83]AUGdUGGUGGUGdAAGGGUUGUUGGGdUGdUCGACGCA 3′(see NOX-D19001-D09-17-30-32-40); or l) [SEQ ID NO: 84]5′ AUGdUGGUGGUdGdAAGGGUUGUUGGGdUGdUCGACGCA 3′ (see NOX-D19001-D09-16-17-30-32-40 = NOX-D19001-6xDNA),

wherein G, A, U and C are ribonucleotides, and dG, dA and dU are2′-deoxyribonucleotides.

The binding affinity of C5a binding nucleic acid molecule NOX-D19001 wassignificantly improved by replacing one up to six ribonucleotides by2′-deoxyribonucloetides as determined by surface plasmon resonancemeasurement and exemplarily shown for the C5a binding nucleic acidsNOX-D19001-D09 and NOX-D19001-D09-16-17-30-32-40 (also referred to asNOX-D19001-6×DNA) (FIG. 6):

NOX-D19001: K_(D) of 1.38 nM,

NOX-D19001-D09: K_(D) of 709 pM,

NOX-D19001-D09-16-17-30-32-40: K_(D) of 361 pM.

NOX-D19001-6×DNA comprises a central stretch of nucleotides with five2′-deoxyribonucleotides instead of ribonucleotides, a first terminalstretch of nucleotides with five ribonucleotides and a second terminalstretch of nucleotides with four ribonucleotides and one2′-deoxyribonucleotide. Surprisingly, the inventors could show that thefirst and the second terminal stretch of nucleotides can be truncatedwithout reduction in affinity to four or three nucleotides. As shownherein, the first and the second terminal stretch of nucleotides ofNOX-D19001-6×DNA could be truncated from five to three nucleotides (seeNOX-D19001-6×DNA-020 also referred to as NOX-D20001) while retainingaffinity (FIG. 4A).

FIG. 4 demonstrates the successful combination ofribonucleotide-to-2′-deoxyribonucleotide substitution and truncation:The mother molecule of NOX-D19001-6×DNA and NOX-D19001-6×DNA-020 (alsoreferred to as NOX-D20001), NOX-D19001, consisting of ribonucleotidesand a first and a second terminal stretch of nucleotides with fivenucleotides each has a binding affinity (K_(D)) of 1.38 nM. After sixribonucleotide-to-2′-deoxyribonucleotide substitutions (leading toNOX-D19001-6×DNA) and truncation to a first and a second terminalstretch of nucleotides with three nucleotides (leading toNOX-D19001-6×DNA-020, also referred to as NOX-D20001) the bindingaffinity for human C5a was improved by a more than factor four(NOX-D20001, K_(D) of 0.3 nM). Truncation of the first or the secondstretch of nucleotides to one nucleotide led to reduced activity, butsuch molecules still bind to C5a with K_(D)'s lower than 10 nM (see FIG.4A, 4B)

Another example for the successful substitution of ribonucleotides by2′-deoxyribonucleotides is shown in FIG. 5. Molecule NOX-D19001-020 is atruncated derivative of NOX-D19001 and has a K_(D) of 11.3 nM (see FIG.5) instead of 1.38 nM as determined for NOX-D19001 (see FIGS. 1 and 2).Both molecules comprise the identical central stretch ofribonucleotides, but NOX-D19001-020 comprises of a first terminalstretch of only three instead of five ribonucleotides and a secondterminal stretch of only three instead of five ribonucleotides. Bysubstitution of two or three ribonucleotides by 2′-deoxyribonucleotidesin the central stretch of nucleotides and optionally of oneribonucleotide by 2′-deoxyribonucleotide in the second terminal stretchof nucleotides the binding affinity of NOX-D19001-020 can be improved bya factor of more than 10 (see FIG. 5, NOX-D19001-2×DNA-020,NOX-D19001-3×DNA-020, NOX-D19001-2dU-1 dC-020 also referred to asNOX-D21001, NOX-D19001-3dU-1dC-020).

Taken together, the first and the second terminal stretches of C5abinding nucleic acid molecules comprise one, two, three, four or fivenucleotides (FIG. 1 to FIG. 5), whereby the stretches optionallyhybridize with each other, whereby upon hybridization a double-strandedstructure is formed. This double-stranded structure can consist of oneto five basepairs. However, such hybridization is not necessarily givenin the molecule.

Analyzing the first terminal stretch of nucleotides and the secondterminal stretch of nucleotides of all tested C5a binding nucleic acidmolecules the generic formula for the first terminal stretch ofnucleotides is 5′ Z₁Z₂Z₃Z₄G 3′ and the generic formula for the secondterminal stretch of nucleotides is 5′ Z₅Z₆Z₇Z₈ Z₉ 3′,

wherein

Z₁ is G or absent, Z₂ is S or absent, Z₃ is S or absent, Z₄ is B orabsent, Z₅ is C or dC, Z₆ is V or absent, Z₇ is S or absent, Z₈ is S orabsent, Z₉ is C or absent, and

G, S, B, C, V are ribonucleotides, and dC is a 2′-deoxyribonucleotide,

whereby in a first preferred embodiment

-   -   a) Z₁ is G, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is V,        Z₇ is S, Z₈ is S, Z₉ is C, or    -   b) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   c) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   d) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is absent, Z₈ is absent, Z₉ is absent, or    -   e) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   f) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   g) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   h) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is absent, Z₅ is        C or dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is C, or    -   i) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   j) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   k) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is absent, Z₅ is        C or dC, Z₆ is V, Z₇ is S, Z₈ is S, Z₉ is absent, or    -   l) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   m) Z₁ is absent, Z₂ is S, Z₃ is S, Z₄ is B, Z₅ is C or dC, Z₆ is        V, Z₇ is absent, Z₈ is absent, Z₉ is absent, or    -   n) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is absent, Z₅ is        C, Z₆ is V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   o) Z₁ is absent, Z₂ is absent, Z₃ is absent, Z₄ is B, Z₅ is C or        dC, Z₆ is V, Z₇ is S, Z₈ is absent, Z₉ is absent, or    -   p) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is V, Z₇ is absent, Z₈ is absent, Z₉ is absent, or    -   q) Z₁ is absent, Z₂ is absent, Z₃ is S, Z₄ is B, Z₅ is C or dC,        Z₆ is absent, Z₇ is absent, Z₈ is absent, Z₉ is absent;

in a second preferred embodiment

-   -   a) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCCUG 3′ and the second terminal        stretch of nucleotides comprises a nucleotide sequence 5′ CAGGC        or    -   b) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCCUG 3′ and the second terminal        stretch of nucleotides comprises a nucleotide sequence of 5′        dCAGGC 3′, or    -   c) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ CCUG 3′ or 5′ CUG 3′ or 5′ UG 3′ or 5′        G 3′, and the second terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ dCAGGC 3′, or    -   d) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCUG 3′ and the second terminal        stretch of nucleotides comprises a nucleotide sequence of 5′        dCAGC 3′, or    -   e) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCCG 3′ and the second terminal        stretch of nucleotides comprises a nucleotide sequence of 5′        dCGGC 3′, or    -   f) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GGCG 3′ and the second terminal        stretch of nucleotides comprises a nucleotide sequence of 5′        dCGCC 3′, or    -   g) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ CUG 3′ or 5′ UG 3′ or 5′ CG 3′ or 5′ G        3′, and the second terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ dCAGC 3′, or    -   h) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCUG 3′, and the second terminal        stretch of nucleotides comprises a nucleotide sequence of 5′        dCAC 3′ or 5′ dCC 3′ or 5′ dCA 3′, or    -   i) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GUG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCAC 3′, or    -   j) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ UG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCA 3′, or    -   k) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCGC 3′, or    -   l) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ CG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCGC 3′, or    -   m) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ G 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCGC 3′, or    -   n) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCC 3′, or    -   o) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dC 3′, or    -   p) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ dCC 3′, or    -   q) the first terminal stretch of nucleotides comprises a        nucleotide sequence of 5′ GCG 3′ and the second terminal stretch        of nucleotides comprises a nucleotide sequence of 5′ CGC 3′.

In order to prove their functionality, the C5a binding nucleic acidmolecules NOX-D19001, NOX-D20001 and NOX-D21001 were synthesized as aSpiegelmer comprising an amino-group at its 5′-end. To saidamino-modified Spiegelmers a 40 kDa PEG-moiety was coupled leading toC5a binding Spiegelmers NOX-D19, NOX-D20 and NOX-D21. Synthesis andPEGylation of the Spiegelmer is described in Example 2.

The effect of improved binding affinity could be shown for thefunctionality of C5a binding nucleic acid molecules. As determined by achemotaxis assay (Example 5), the C5a binding nucleic acid moleculeNOX-D19 (IC₅₀=1.9 nM) exclusively consisting of ribonucleotides was lesspotent to inhibit the function of human C5a than the NOX-D20, aderivative C5a binding nucleic acid molecule of NOX-D19 comprising sixribonucleotide-to-2′-deoxyribonucleotide substitutions (IC₅₀=0.28 nM)(FIG. 7).

NOX-D20 showed a very high affinity binding to murine C5a with adissociation constant K_(D) of 19 pM, whereas for human C5a a K_(D) of299 pM was determined (Example 4, FIG. 8). NOX-D20 inhibits the functionof human C5a with an inhibitory constant IC₅₀ of 275 pM as determined bya chemotaxis assay (Example 5, FIGS. 7 and 12 A). For stoichiometricreasons, the sensitivity of the chemotaxis assays for mouse C5a islimited to 150 pM due a stimulatory concentration of mouse C5a of 300pM. Accordingly, for mouse C5a an IC₅₀ of 140 pM was measured forNOX-D20 (Example 5, FIG. 12 A).

NOX-D20 showed no binding to C5a from rat or rhesus monkey, indicatingvery high target specificity (FIG. 8). From the polypeptide sequencealignment of human, mouse, rat and rhesus monkey C5a and the determinedspecificity it is most likely that the residues Serine16 and Valine28 ofhuman C5a are essential binding residues on C5a (FIG. 11). These areconserved in human and murine C5a but are different in rhesus monkey andrat C5a.

NOX-D21 contains the major affinity-improving sites of NOX-D20 andshowed a high affinity to human and murine C5a as shown by Biacoremeasurement (K_(D)(murine C5a)=29 pM, K_(D)(human C5a)=815 pM,K_(D)(human C5)=413 pM, see FIG. 11). NOX-D21 inhibits the function ofhuman C5a with an inhibitory constant IC₅₀ of 476 pM, as determined by achemotaxis assay (Example 5, FIG. 12B).

In vivo a truncated version of C5a is generated by enzymatic cleavage ofthe C-terminal arginine residue, known as des-Arg-C5a (also referred toas C5a_(desArg)). The biological function of des-Arg-C5a is not fullyunderstood but there is evidence that des-Arg-C5a retains leukocyteactivating functions. Therefore it was investigated, whether NOX-D20also bound to des-Arg-C5a. NOX-D20 showed a dose-dependent binding toimmobilized recombinant human des-Arg-C5a (FIG. 9).

Detailed kinetic evaluation as described showed that human des-Arg-C5ais bound by NOX-20 with comparable affinity to the full-length human C5awith a dissociation constant of 316 pM and 299 pM, respectively. NOX-D21bound to mouse and human des-Arg-C5a with dissociation constants of 28pM and 854 pM, respectively (FIG. 10). Thus even after cleavage of C5ato des-Arg-C5a C5a binding nucleic acid molecules such as NOX-20 andNOX-D21 still bind to their target.

Surprisingly NOX-D20 and NOX-D21 also showed binding to C5 purified fromhuman plasma with an affinity of 164 pM and 413 pM, respectively (FIGS.9 and 10). This phenomenon could not be foreseen. However, it isplausible since C5a is a part of C5 that is cleaved off by the C5convertase when the complement system is activated or by thrombin orother members of an activated coagulation system. Furthermore, the C5purified from human plasma carries the native glycosylation structure onasparagine64. Glycosylation had not been present on the murine mirrorimage C5a polypeptide that was used for identification of NOX-D19,NOX-D20, NOX-D21 and other nucleic acid molecules according to thepresent invention.

Binding to C5 may influence pharmacokinetics due to the expected lowclearance of the large C5 protein and a published plasma concentrationof 350-390 nM. Binding to C5 may also influence pharmacodynamics. C5 isbound by C5a binding nucleic acid molecules such as NOX-20 and NOX-D21and thus C5a is already blocked by the Spiegelmer before it is liberatedand may lead to receptor signaling.

EXAMPLE 2: SYNTHESIS AND DERIVATIZATION OF APTAMERS AND SPIEGELMERS

Small Scale Synthesis

Aptamers (D-RNA nucleic acids) and Spiegelmers (L-RNA nucleic acids)were produced by solid-phase synthesis with an ABI 394 synthesizer(Applied Biosystems, Foster City, Calif., USA) using 2′TBDMS RNAphosphoramidite chemistry (Damha and Ogilvie, 1993). rA(N-Bz)-, rC(Ac)-,rG(N-ibu)-, and rU-phosphoramidites in the D- and L-configuration werepurchased from ChemGenes, Wilmington, Mass. Aptamers and Spiegelmerswere purified by gel electrophoresis.

Large Scale Synthesis Plus Modification

Spiegelmers were produced by solid-phase synthesis with an ÄktaPilot100synthesizer (Amersham Biosciences; General Electric Healthcare,Freiburg) using 2′TBDMS RNA phosphoramidite chemistry (Damha andOgilvie, 1993). L-rA(N-Bz)-, L-rC(Ac)-, L-rG(N-ibu)-, andL-rU-phosphoramidites were purchased from ChemGenes, Wilmington, Mass.The 5′-amino-modifier was purchased from American InternationalChemicals Inc. (Framingham, Mass., USA). Synthesis of the unmodified or5′-Amino-modified Spiegelmer was started on L-riboG, L-riboC, L-riboA orL-riboU modified CPG pore size 1000 Å (Link Technology, Glasgow, UK. Forcoupling (15 min per cycle), 0.3 M benzylthiotetrazole (CMS-Chemicals,Abingdon, UK) in acetonitrile, and 3.5 equivalents of the respective 0.1M phosphoramidite solution in acetonitrile was used. Anoxidation-capping cycle was used. Further standard solvents and reagentsfor oligonucleotide synthesis were purchased from Biosolve(Valkenswaard, NL). The Spiegelmer was synthesized DMT-ON; afterdeprotection, it was purified via preparative RP-HPLC (Wincott et al.,1995) using Source15RPC medium (Amersham). The 5′DMT-group was removedwith 80% acetic acid (30 min at RT). Subsequently, aqueous 2 M NaOAcsolution was added and the Spiegelmer was desalted by tangential-flowfiltration using a 5 K regenerated cellulose membrane (Millipore,Bedford, Mass.).

PEGylation of Spiegelmers

In order to prolong the Spiegelmer's plasma residence time in vivo,Spiegelmers was covalently coupled to a 40 kDa polyethylene glycol (PEG)moiety at 5′-end

For PEGylation (for technical details of the method for PEGylation seeEuropean patent application EP 1 306 382), the purified 5′-aminomodified Spiegelmer was dissolved in a mixture of H₂O (2.5 ml), DMF (5ml), and buffer A (5 ml; prepared by mixing citric acid.H₂O [7 g], boricacid [3.54 g], phosphoric acid [2.26 ml], and 1 M NaOH [343 ml] andadding water to a final volume of 1 l; pH=8.4 was adjusted with 1 MHCl).

The pH of the Spiegelmer solution was brought to 8.4 with 1 M NaOH.Then, 40 kDa PEG-NHS ester (Jenkem Technology, Allen, Tex., USA) wasadded at 37° C. every 30 min in six portions of 0.25 equivalents until amaximal yield of 75 to 85% was reached. The pH of the reaction mixturewas kept at 8-8.5 with 1 M NaOH during addition of the PEG-NHS ester.

The reaction mixture was blended with 4 ml urea solution (8 M), and 4 mlbuffer B (0.1 M triethylammonium acetate in H₂O) and heated to 95° C.for 15 min. The PEGylated Spiegelmer was then purified by RP-HPLC withSource 15RPC medium (Amersham), using an acetonitrile gradient (bufferB; buffer C: 0.1 M triethylammonium acetate in acetonitrile). Excess PEGeluted at 5% buffer C, PEGylated Spiegelmer at 10-15% buffer C. Productfractions with a purity of >95% (as assessed by HPLC) were combined andmixed with 40 ml 3M NaOAc. The PEGylated Spiegelmer was desalted bytangential-flow filtration (5 K regenerated cellulose membrane,Millipore, Bedford Mass.).

EXAMPLE 3: DETERMINATION OF BINDING CONSTANTS TO C5A FOR APTAMERS(PULL-DOWN ASSAY)

Direct Pull-Down Assay

The affinity of C5a binding nucleic acids was measured as binding ofaptamers (D-RNA nucleic acids) to biotinylated mouse D-C5a (SEQ.ID. 89)in a pull down assay format at 37° C. Aptamers were 5′-phosphate labeledby T4 polynucleotide kinase (Invitrogen) using [γ-³²P]-labeled ATP(Hartmann Analytic, Braunschweig, Germany). The specific radioactivityof labeled aptamers was 200,000-800,000 cpm/pmol. Assays were carriedout in selection buffer (20 mM Tris-HCl pH 7.4; 150 mM NaCl; 5 mM KCl; 1mM MgCl₂; 1 mM CaCl₂; 4 U/ml RNase inhibitor (RNaseOUT, Invitrogen);0.1% [w/vol] Tween-20 supplemented with 50 μg/ml bovine serum albumin(Sigma), and 10 μg/ml non-specific Spiegelmer in order to preventadsorption of binding partners with surfaces of used plasticware or theimmobilization matrix). Aptamers were incubated after de- andrenaturation at 0.2-1 nM concentration at 37° C. in selection buffertogether with varying amounts of biotinylated mouse D-C5a for 3-4 hoursin order to reach equilibrium at low concentrations. The concentrationrange of biotinylated mouse D-C5a was set from 640 pM to 10 μM; totalreaction volume was 80-200 μl. Biotinylated mouse D-C5a and complexes ofaptamer and biotinylated mouse D-C5a were immobilized on 5 μlNeutrAvidin Agarose Plus particles (Pierce Biotechnology) which had beenpreequilibrated with selection buffer. Particles were kept in suspensionfor 30 min at the 37° C. in a thermomixer. Immobilized radioactivity wasquantitated in a scintillation counter after detaching the supernatantand appropriate washing. The percentage of binding was plotted againstthe concentration of biotinylated mouse D-C5a and dissociation constantswere obtained by using software algorithms (GraphPad Prism) assuming a1:1 stoichiometry.

Competitive Pull-Down Assay

In order to compare different D-C5a binding nucleic acids, a competitiveranking assay was performed. For this purpose the most affine aptameravailable was radioactively labeled (see above) and served as reference.After de- and renaturation it was incubated in selection buffer at 37°C. with biotinylated mouse D-C5a at conditions that resulted in around5-10% binding to the biotinylated mouse D-C5a after immobilization andwashing on 4 μl NeutrAvidin Agarose Plus particles (PierceBiotechnology) without competition. An excess of de- and renaturednon-labeled D-RNA aptamer variants was added at concentrations rangingfrom 9 pM-400 nM with the labeled reference aptamer to parallel bindingreactions; total reaction volume was 160-400 μl. After 3-4 hourincubation biotinylated mouse D-C5a and complexes of aptamer andbiotinylated were immobilized and assays were analysed as describedabove. The aptamers to be tested competed with the reference aptamer fortarget binding, thus decreasing the binding signal in dependence oftheir binding characteristics. The aptamer that was found most active inthis assay could then serve as a new reference for comparative analysisof further aptamer variants.

EXAMPLE 4: BIACORE MEASUREMENT OF SPIEGELMERS BINDING TO C5A AND RELATEDPEPTIDES

The instrument was set to an enduring temperature of 37° C. The Biacore2000 instrument was cleaned using the DESORB method before the start ofeach experiment/immobilization of a new chip. After docking amaintenance chip, the instrument was consecutively primed with desorbsolution 1 (0.5% sodium dodecyl sulphate, SDS), desorb solution 2 (50 mMglycine, pH 9.5) and HBS-EP buffer. Finally, the system was primed withHBS-EP buffer.

For Biacore experiments the C5a-binding Spiegelmers were prepared insterile water and had a concentration of 100

The CM5 chip was primed with HBS-EP buffer and equilibrated until astable baseline was observed. The flow cells were immobilized beginningfrom flow cell 4 to flow cell 1. 100 μl of a 1:1 mixture of 0.4 M EDCand 0.1 M NHS were injected using the QUICKINJECT command at a flow of10 μl/min. Activation of the flow cell was monitored by an increase inRU after NHS/EDC injection (typically 150-500 RU for CM5 chips).Solutions of 0.1-1 μg/ml in 10 mM NaAc pH5.5 for C5a or 10 mM NaAc pH5.5for human C5 were transferred to a vial and injected using theMANUALINJECT command at a flow of 10 μl/min. 1000-3000 RU wereimmobilized the chip. All flow cells were then blocked with an injectionof 70 μl of 1 M ethanolamine hydrochloride, pH 8 at a flow of 10 μl/min.Injection of 30 μl of the regeneration solution (1 M NaCl) at a flow of30 μl/min was performed to remove unspecifically bound protein from thechip surface.

Kinetic parameters and dissociation constants were evaluated by a seriesof Spiegelmer injections at concentrations of2,000−1,000−500−200−125−62.5−31.3−15.6(2×)−7.8−3.9−1.95−0.98−0.48−0.24−0.12−0 nM diluted in running buffer,starting with the lowest concentration. In all experiments, the analysiswas performed at 37° C. using the Kinject command defining anassociation time of 240 and a dissociation time of 240 seconds at a flowof 30 μl/min. The assay was double referenced, whereas FC1 served as(blocked) surface control (bulk contribution of each Spiegelmerconcentration) and a series of buffer injections without analytedetermined the bulk contribution of the buffer itself. At least oneSpiegelmer concentration was injected twice to monitor the regenerationefficiency and chip integrity during the experiments. Regeneration wasperformed by injecting 60 μl of 1M NaCl at a flow of 30 μl/min.Stabilization time of baseline after each regeneration cycle was set to1 min at 30 μl/min.

Data analysis and calculation of dissociation constants (K_(D)) was donewith the BIAevaluation 3.1.1 software (BIACORE AB, Uppsala, Sweden)using a modified Langmuir 1:1 stoichiometric fitting algorithm, with aconstant RI and mass transfer evaluation with a mass transportcoefficient kt of 1×10⁷ [RU/M*s].

EXAMPLE 5: DETERMINATION OF INHIBITORY CONCENTRATION IN A CHEMOTAXISASSAY

Generation of a Cell Line Expressing the Human Receptor for C5a

A stably transfected cell line expressing the human receptor for C5a wasgenerated by transfecting BA/F3 mouse pro B cells with a plasmid codingfor the human C5a receptor (NCBI accession NM_001736 in pcDNA3.1+).Cells expressing C5aR were selected by treatment with geneticin andtested for expression with RT-PCR and for functionality with chemotaxisassay.

Chemotaxis Assay

The day before the experiment, cells are seeded in a new flask at0.3×10⁶/ml. For the experiment, cells were centrifuged, washed once inHBH (HBSS, containing 1 mg/ml bovine serum albumin and 20 mM HEPES) andresuspended at 1.33×10⁶ cells/ml. 75 μl of this suspension were added tothe upper compartments of a 96 well Corning Transwell plate with 5 μmpores (Costar Corning, #3388; NY, USA). In the lower compartmentsrecombinant human C5a (SEQ.ID. 50) or mouse C5a (SEQ.ID. 54) waspre-incubated together with Spiegelmers in various concentrations in 235μl HBH at 37° C. for 20 to 30 min prior to addition of cells. Cells wereallowed to migrate at 37° C. for 3 hours. Thereafter the insert plate(upper compartments) was removed and 30 μl of 440 μM resazurin (Sigma,Deisenhofen, Germany) in phosphate buffered saline was added to thelower compartments. After incubation at 37° C. for 2.5 hours,fluorescence was measured at an excitation wavelength of 544 nm and anemission wavelength of 590 nm.

Fluorescence values are corrected for background fluorescence (no C5a inwell) and plotted against Spiegelmer concentration. The IC₅₀ values aredetermined with non-linear regression (4 parameter fit) using GraphPadPrism. Alternatively, the value for the sample without Spiegelmer (C5aonly) is set 100% and the values for the samples with Spiegelmer arecalculated as percent of this. The percent-values are plotted againstSpiegelmer concentration and the IC₅₀-values are determined as describedabove.

Determination of the Half-Maximal Effective Concentration (EC₅₀) forHuman and Mouse C5a

After 3 hours migration of BA/F3/huC5aR cells towards various human C5aor mouse C5a concentrations, dose-response curves for human and mouseC5a were obtained, indicating half effective concentrations (ECH) of 0.1nM for huC5a and 0.3 nM for mC5a. For the experiments on inhibition ofchemotaxis by Spiegelmers 0.1 nM human C5a and 0.3 nM mouse C5a wereused.

EXAMPLE 6: INHIBITION OF C5A-INDUCED ACTIVATION OF PRIMARY HUMANNEUTROPHILS

Isolation of Human PMNs

Polymorphonuclear leukocytes (PMN) were isolated from whole blood bydiscontinuous gradient centrifugation at room temperature. Blood wascollected in acid citrate dextrose containing blood collection tubes(Sarstedt). Dextran 500 (Accurate Chemical) was added to a finalconcentration of 2% w/v and the blood/dextran was layered on toHistopaque (1.077 g/ml, Sigma). After centrifugation all liquid andcells above the gradient interface were discarded. Pellet and circa 80%of remaining liquid above were collected and diluted 1:1 with a mixtureof Voluven 80% v/v (Fresenius Kabi), PBS 16% v/v (Sigma) and ACD 4% v/v(Sigma). Mixture was centrifuged at 400 rpm for 15 minutes. Supernatantwas collected and centrifuged at 1,000 rpm for 7 minutes. The pellet wasgently re-suspended and remaining erythrocytes were removed by lysis.

Inhibition of C5a-Induced Chemotaxis of Human PMNs

Human C5a (1 nM) was preincubated with indicated concentrations ofNOX-D19 or NOX-D20 in HBSS+0.01% BSA+25 mM HEPES in the lower chamber ofa chemotaxis plate. Human neutrophils were added to the upper chambes ofa chemotaxis plate and chemotaxis was performed over 25 min at 37° C.and 5% CO₂. Following incubation the upper chamber was fitted to a whiteluminescence plate containing Accutase to harvest cells bound to theunderside of the chemotaxis mesh. Glo reagent (Promega) was added andequilibrated for 10 min. Luminescence was measured using a BiotekSynergy 2 plate reader.

Inhibition of C5a-Induced Elastase Release by Human PMNs

Human neutrophils were primed with TNFα (10 ng/ml) and cytochalasin B (5μg/ml) for 30 minutes at 37° C., 5% CO₂. Cells were stimulated for 45min with human C5a (30 nM) which had been pre-incubated with NOX-D19 orNOX-D20 at indicated concentrations. Cells were then separated bycentrifugation and 25 μl of supernatant were incubated with elastasesubstrate (Calbiochem) in Tris-HCl 0.1 M pH 7.4 for 1 h at 37° C. withreadings being taken at an absorbance of 405 nm every 5 minutes. Thekinetic data was analysed to determine the v_(max) for each sample. Themean percentage elastase activity relative to control was calculated foreach sample (background not subtracted).

Results

NOX-D19 and NOX-D20 efficiently inhibit the activation of freshlyisolated human peripheral blood PMN by C5a. 10 nM NOX-D19 or NOX-D20were sufficient to block more than 85% of huC5a-induced chemotaxis ofhuman PMN (FIG. 13 A). HuC5a-induced release of antimicrobial elastasewas efficiently inhibited by NOX-D19 and NOX-D20 (FIG. 13 B). 30 nMNOX-D19 or NOX-D20 suppressed about 50% of C5a-induced elastase release.Of note, for stoichiometric reasons the sensitivity of this assay islimited to IC₅₀=15 nM, as elastase release is induced by 30 nM huC5a.

EXAMPLE 7: C5A BINDING NUCLEIC ACIDS DO NOT INTERFERE WITHCOMPLEMENT-DEPENDENT HEMOLYSIS

The ultimate product of the complement cascade is the membrane attackcomplex (MAC), a pore consisting of C5b-9. MAC is believed to insertinto the cytoplasmic membranes of pathogens and kill them by inductionof cytoplasmic leakage.

The C5a binding nucleic acids (Spiegelmers) presented here have beenshown to recognize C5a in the context of C5 (see Example 1, FIG. 9 andFIG. 10). Therefore it was investigated whether C5 cleavage to theanaphylatoxin C5a and C5b, which is part of the MAC is inhibited bythese the Spiegelmers. This was achieved by using a complement-dependentsheep erythrocyte hemolysis test.

Methods

Reconstituted human lyophilized serum (‘Human Complement Serum’ (SigmaAldrich, Germany) was pre-incubated with PEGylated Spiegelmers NOX-D19,NOX-D20 and NOX-D21 in the range of 10 nM to 10,000 nM in 96-well plates(Nunc-Immuno™ Plate, MaxiSorp Surface™). As a positive control theC5-binding aptamer C5C6 with maximal 2′OMe purine and 2′fluoropyrimidine substitution (Biesecker et al. 1999) (synthesized in house)which inhibits C5 cleavage was used in the same concentration range. Asa control for potential unspecific Spiegelmer effects on the assayPEGylated Spiegelmers with the reverse sequence of NOX-D19 and NOX-D21,revNOX-D19 and revNOX-D21 were included. revNOX-D19 and revNOX-D21 wereearlier shown not to inhibit C5a in a Biacore and cell based assays.After 1 hour incubation at 37° C. sheep erythrocytes opsonized withrabbit anti-sheep erythrocyte antibodies, known as hemolytic system(Institut Virion/Serion GmbH, Germany) were added to the pre-incubatedserum complement inhibitor mixture. Complement is activated via theclassical pathway leading to the cleavage of C5 to C5a and C5b. C5b thenassociates with C6-C9 to form the lytic membrane attack complex (MAC).Sheep erythrocyte hemolysis due to MAC formation was determined 30 minlater by a colorimetric measurement after spinning down intact cells.The higher the degree of hemolysis the higher the absorption at 405 nm(measured in a Fluo Star plate reader).

Results

The aptamer C5C6 inhibited complement-dependent lysis of the sheeperythrocytes with an IC₅₀ of approximately 1 μM (FIG. 14 A, B). TheSpiegelmers tested, namely C5 and C5a binding nucleic acids NOX-D19 andNOX-D20 (FIG. 14 A) and NOX-D21 (FIG. 14 B) and the non-C5- orC5a-binding Spiegelmers revNOX-D19 (FIG. 14 A) and revNOX-D21 (FIG. 14B) did not inhibit hemolysis.

Discussion

The C5a binding Spiegelmers tested were shown not to inhibit MACformation and are therefore selective antagonists of C5a only. If usedas a medicine, this may be advantageous, since inhibition ofMAC-formation can compromise the body's defense mechanism to invadingpathogens, mainly Gram-negative bacteria.

EXAMPLE 8: THE C5A-BINDING NUCLEIC ACID NOX-D19 SHOWS EFFICACY IN THEMURINE CECAL LIGATION AND PUNCTURE MODEL FOR POLYMICROBIAL SEPSIS

The effect of intraperitoneal injections of NOX-D19 on the course ofpolymicrobial sepsis was tested in a rodent cecal ligation and puncture(CLP) model.

Methods

Animal Model

10-12 week old male C57BL/6 mice (Charles River Laboratories, Germany)were used for the study. Peritonitis was surgically induced under lightisofluran anesthesia. Incisions were made into the left upper quadrantof the peritoneal cavity (normal location of the cecum). The cecum wasexposed and a tight ligature was placed around the cecum with suturesdistal to the insertion of the small bowel (75% were ligated). Onepuncture wound was made with a 24-gauge needle into the cecum and smallamounts of cecal contents were expressed through the wound. The cecumwas replaced into the peritoneal cavity and the laparotomy site wasclosed. 500 μl saline was given s.c. as fluid replacement. Sham animalsunderwent the same procedure except for ligation and puncture of thececum. Finally, animals were returned to their cages with free access tofood and water.

Study Groups

4 groups (n=6 mice for sham surgery and n=10 mice per group for CLPsurgery) were tested: (1) sham surgery with vehicle (saline) treatment,(2) CLP surgery with vehicle treatment, (3) CLP surgery with low doseNOX-D19 (1 mg/kg) treatment and (4) CLP surgery with high dose NOX D19(10 mg/kg) treatment. The investigators were blinded to the treatmentstrategy and did not know which compound contains vehicle or verum.Route of administration was i.p. every day for 6 days starting at timeof the CLP surgery.

Survival

Follow up was 7 days in each group. Mice were monitored daily and KaplanMeier survival curves were generated using GraphPad Prism 4 software.

Blood Drawing

Blood samples were obtained under light ether anaesthesia from thecavernous sinus with a capillary prior to surgery (baseline, day −4) andat day 1 after surgery to allow measurement of routine serum markers ofacute kidney injury (serum creatinine, and blood urea nitrogen, BUN) andacute liver failure (serum alanin-aminotransferase, serum ALT). Thelevel of aspartate aminotransferase (serum AST) was measured in serum asa marker of multiorgan failure. Measurement of clinical chemistryparameters was performed on an Olympus analyser (AU400).

Statistics

Statistical significance was calculated by Student's T-test. Forsurvival Kaplan Meier curves were generated and log rank test forsignificance was performed. GraphPad Prism 4 software was used.

Results

Survival

As expected no mortality occurred in animals with sham surgery withoutCLP (FIG. 15). In mice that received CLP surgery and were treated withvehicle only, median survival was 1.5 days NOX-D19 treatment after CLPsurgery improved median survival (FIG. 15). Mice treated with low doseNOX-D19 (1 mg/kg) showed the longest median survival (5 days, p<0.0001vs. vehicle). Mice treated with high dose NOX-D19 (10 mg/kg) had amedian survival of 3 days which was significantly longer than in vehicletreated mice (p=0.0401) but was not significantly different from lowdose NOX-D19 treatment (p=0.4875). 100% of vehicle mice died within 4days after CLP surgery. 100% and 90% mortality occurred not before 7days in mice treated with low and high dose NOX-D19, respectively (FIG.15).

Clinical Chemistry

Renal Function

The serum creatinine and blood urea nitrogen (BUN) concentration areparameters for renal function. Renal function was assessed before thestart of the study (day −4) and on day 1 after CLP surgery.

By day 1 CLP induced a significant increase in serum creatinine levelsin vehicle treated mice. Low dose NOX-D19 treatment (1 mg/kg) preventedthis increase (FIG. 16 A). In mice treated with high dose NOX-D19 (10mg/kg) a moderate but statistically not significant increase in serumcreatinine levels was observed (p=0.1873 vs. vehicle) (FIG. 16 A).

BUN (FIG. 16 B), which is a more sensitive parameter of renal functionthan creatinine, was significantly increased at day 1 after CLP surgeryin vehicle treated mice. Treatment of mice with low and high doseNOX-D19 significantly suppressed the increase of BUN upon CLP (FIG. 16B).

Liver Function

The most reliable marker of hepatocellular injury or necrosis is serumalanine aminotransferase (serum ALT). All groups showed an increase ofserum ALT at day 1 after CLP surgery. However, both groups of NOX-D19treated septic mice demonstrated improved liver function compared tovehicle treated mice (FIG. 17 A).

Multiorgan Failure

The serum level of aspartate aminotransferase (serum AST) (FIG. 17 B)was measured as a marker of multiorgan failure since AST has been shownto be elevated in diseases affecting other organs besides liver, such asmyocardial infarction, acute pancreatitis, acute hemolytic anemia,severe burns, acute renal disease, musculoskeletal diseases, and trauma.

Similar to ALT, all groups showed an increase of AST levels at day 1after CLP surgery (p<0.001 vs. sham). However, similar to liverfunction, both groups of NOX-D19 treated septic mice demonstrated lesspronounced AST levels compared to vehicle treated mice (FIG. 17 B).

EXAMPLE 9: THE IMPROVED C5A-BINDING NUCLEIC ACID NOX-D20 SHOWS EFFICACYIN THE MURINE CECAL LIGATION AND PUNCTURE MODEL FOR POLYMICROBIAL SEPSIS

The effect of intraperitoneal injections of NOX-D20 on the course ofpolymicrobial sepsis was tested in a rodent cecal ligation and puncture(CLP) model.

Methods

Animal Model

Polymicrobial sepsis was induced in 10-12 week old male C57BL/6 mice(Charles River Laboratories, Germany) as described in Example 8 with60-75% of the cecum being ligated.

Survival

Follow up was 7 days in each group. Mice were monitored daily and KaplanMeier survival curves were generated using GraphPad Prism 4 software.

Study Groups

5 groups (n=5 mice for sham surgery and n=10 mice per group for CLPsurgery) were tested: (1) sham surgery with vehicle (saline) treatment,(2) CLP surgery with vehicle treatment, (3) CLP surgery with daily lowdose NOX-D20 (1 mg/kg) treatment, (4) CLP surgery with daily high doseNOX-D20 (3 mg/kg) treatment and (5) CLP surgery with a single low doseNOX-D20 (1 mg/kg) after surgery followed by daily vehicle treatment. Theinvestigators were blinded to the treatment strategy and did not knowwhich compound contains vehicle or verum. Route of administration wasi.p.

Clinical Chemistry and Inflammatory Parameters

Blood samples were obtained as described in Example 8 under light etheranaesthesia from the cavernous sinus with a capillary at day 1 aftersurgery. Routine serum markers of acute kidney injury (serum creatinine,and blood urea nitrogen, BUN), acute liver failure (serum ALT) andendothelial injury (serum lactate dehydrogenase, serum LDH) weremeasured. Peritoneal lavage (PL) was performed using 3 ml PBS. Thevolume of the collected PL was measured in each sample, and the totalcell count was assessed using a hemocytometer (Neubauer Zaehlkammer,Gehrden, Germany). Serum and PL levels of tumor necrosis factor alpha(TNF-α), interleukin-6 (IL-6), and CCL2 (=macrophage chemoattractantprotein-1, MCP-1) were quantified by bead-based flow cytometry assay(CBA Kit, BD Biosciences, Heidelberg, Germany). Serum and PLconcentrations of CXCL1 (=keratinocyte chemoattractant, KC) and CXCL2(=macrophage inflammatory protein 2, MIP-2) were determined by ELISA(R&D Systems, Wiesbaden, Germany). Differential cell count in the PL wasperformed on hematoxylin and eosin (H&E) stained cytospins (cytospin4,Thermo Scientific).

Capillary Leakage

Immediately after CLP surgery, 0.25% w/v Evans blue (200 μl) wasinjected intravenously. After 18 h mice were sacrificed and PL wasperformed as described above. Concentrations of Evans Blue dye in serumand PL fluids was measured spectrophotometrically at 620 nm. Thefollowing formula was used to correct the optical densities forcontamination with heme pigments: E620 (corrected)=E620 (raw)−(E405(raw)×0.014). Plasma exudation was quantitated as the ratio ofextinction in PL fluid to extinction in plasma.

Statistics

Statistical significance was calculated by one-way ANOVA and Dunnettstest. For survival long rank test for significance was performed.GraphPad Prism 4 software was used.

Results

Survival

As expected no mortality occurred in sham operated mice within 7 daysafter surgery (FIG. 18). In vehicle-treated CLP mice median survival was3 days. Daily treatment of mice with 1 mg/kg NOX-D20 significantlyprolonged median survival to 7 days (p=0.0043 vs. vehicle). An increaseof the dosage to 3 mg/kg NOX-D20 had no additional protective effectwith a similar median survival of 6.5 days (p=0.0092 vs. vehicle).Notably, a single injection of 1 mg/kg NOX-D20 after CLP surgery was aseffective as daily treatment and significantly prolonged median survivalto 6.5 days (FIG. 18). While 100% of vehicle treated mice dies within 5days, 30-40% of NOX-D20 treated mice were still alive at the end of theexperiment at day 7 (FIG. 18).

Organ Function

Systemic inflammation often causes multiple organ failure. Increasedserum levels of creatinine and BUN are parameters for decreasedglomerular filtration rate and kidney failure. Both parameters weresignificantly increased in vehicle treated mice one day after CLPsurgery compared to sham mice. NOX-D20 treatment efficiently preventedthe increase of both markers implying a protective effect of NOX-D20 onrenal function (FIG. 19 A, B). Alanine aminotransferase (ALT) is acommon marker of hepatocellular injury and necrosis and CLP-inducedsepsis was associated with increased of ALT serum levels. NOX-D20treated mice demonstrated significantly reduced levels of serum ALTcompared to vehicle treated mice suggesting improved liver function(FIG. 19 C). Elevated serum levels of lactate dehydrogenase (LDH) occurafter tissue injury and are therefore a general marker of organ failure.The increase in LDH levels provoked by CLP was effectively blocked byNOX-D20 (FIG. 20 A). Breakdown of the endothelial barrier and edemaformation is a common fatal event in sepsis. Sepsis induction resultedin a two-fold increase in relative plasma protein extravasation into theperitoneal cavity in vehicle treated compared to sham operated mice.NOX-D20 treatment significantly inhibited capillary leakage (FIG. 20 B).For all parameters tested here 1 mg/kg NOX-D20 was sufficient tosignificantly improve organ function which is reflected in improvedsurvival of NOX-D20 treated mice.

Inflammation

CLP resulted in a strong local and systemic upregulation ofpro-inflammatory cytokines and chemokines. Blockade of C5a by NOX-D20efficiently reduced the concentrations of TNFα, IL-6, CCL2, CXCL1 andCXCL2 in the peritoneum and in serum at day 1 after CLP. Theup-regulation of these chemokines is associated with a recruitment ofpolymorphonuclear leukocytes (PMN) to the peritoneum. Accordingly,C5a-inhibition by NOX-D20 inhibited the accumulation of PMN in theperitoneal cavity (FIG. 20 C). Similarly, infiltration of monocytes wasblocked by NOX-D20.

EXAMPLE 10: EFFICACY OF NOX-D21 IN A MODEL OF ISCHEMIAREPERFUSION-INDUCED ACUTE KIDNEY INJURY

The effect of NOX-D21 on acute kidney injury (AKI) was tested in arodent model of renal ischemia/reperfusion injury (IRI).

Methods

Animal Model

12-15 week old male C57BL/6 mice (Charles River, Germany) wereanaesthetized using isoflurane via a nose mask and placed supine on aheating table to maintain body temperature around 32° C. Midlineincision was performed and the right and left renal pedicle were clippedwith a micro-aneurysm clip for 30 min. After removal of the clip andsuture of the skin mice were returned to the cages and monitored untilfully awake.

Study Groups

3 groups (n=10 mice per group) were tested: (1) IRI surgery with vehicletreatment, (2) IRI surgery with low dose NOX-D21 (1 mg/kg) treatment,(3) IRI surgery with high dose NOX-D21 (10 mg/kg) treatment. Theinvestigators were blinded to the treatment strategy and did not knowwhich compound contains vehicle or verum. NOX-D21 was given i.v. 1 hprior to surgery at d0 and during the next 3 days (d1-d3) it was giveni.p. once daily.

Survival

Mice were monitored daily for 14 days. Kaplan Meier survival curves weregenerated and significance was determined by log-rank test usingGraphPad Prism 4 software.

Results

Survival was significantly improved by treatment with high NOX-D21 (FIG.21). Low dose NOX-D21 treatment resulted in an evident yet notsatistically significant improvement of survival. In the control grouptreated with vehicle only one mouse survived until day 14. NOX-D21treatment increased the percentage of surviving mice to 45-55% (FIG.21).

REFERENCES

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The features of the present invention disclosed in the specification,the claims, the sequence listing and/or the drawings may both separatelyand in any combination thereof be material for realizing the inventionin various forms thereof.

The invention claimed is:
 1. A method comprising administering to asubject with a respiratory disorder or cancer, a nucleic acid moleculethat binds human C5a, wherein the nucleic acid molecule comprises acentral stretch of nucleotides, wherein the central stretch ofnucleotides comprises a nucleotide sequence of (SEQ ID NO: 61)5′ AUGn₁GGUGKUn₂n₃RGGGHUGUKGGGn₄Gn₅CGACGCA 3′,

wherein n₁ is U or dU, n₂ is G or dG, n₃ is A or dA, n₄ is U or dU, n₅is U or dU, and G, A, U, C, H, K, and R are ribonucleotides, and dU, dGand dA are 2′-deoxyribonucleotides to alleviate a symptom of saidrespiratory disorder or said cancer.
 2. The method of claim 1, whereinthe central stretch of nucleotides consists of ribonucleotides and2′-deoxyribonucleotides.
 3. The method of claim 1, wherein the nucleicacid molecule comprises in 5′→3′ direction a first terminal stretch ofnucleotides, the central stretch of nucleotides and a second terminalstretch of nucleotides, wherein the first terminal stretch ofnucleotides comprises one to five nucleotides, and the second terminalstretch of nucleotides comprises one to five nucleotides.
 4. The methodof claim 1, wherein the nucleic acid molecule binds human C5a and mouseC5a.
 5. The method of claim 1, wherein the nucleic acid moleculecomprises at least one binding moiety which binds human C5a and mouseC5a, wherein such binding moiety consists of L-nucleotides.
 6. Themethod of claim 1, wherein said nucleic acid molecule comprises anL-nucleotide.
 7. The method of claim 1, wherein the nucleic acidmolecule is an L-nucleic acid molecule.
 8. The method of claim 1,wherein the nucleic acid molecule is an antagonist of an activitymediated by human and/or mouse C5a.
 9. The method of claim 1, whereinthe nucleic acid molecule comprises a modification group.
 10. The methodof claim 9, wherein the modification group is polyethylene glycol,linear polyethylene glycol, branched polyethylene glycol, hydroxyethylstarch, a peptide, a protein, a polysaccharide, a sterol,polyoxypropylene, polyoxyamidate or poly (2-hydroxyethyl)-L-glutamine.11. The method of claim 10, wherein said linear polyethylene glycol orsaid branched polyethylene glycol comprises a molecular weight of fromabout 20,000 to about 120,000 Da, from about 30,000 to about 80,000 Daor about 40,000 Da.
 12. The method of claim 10, wherein saidhydroxyethyl starch comprises a molecular weight of from about 50 toabout 1000 kDa, from about 100 to about 700 kDa or from 200 to 500 kDa.13. The method of claim 10, wherein said modification group is coupledto the nucleic acid molecule via a linker.
 14. The method of claim 10,wherein the modification group is coupled to the 5′-terminal nucleotideand/or the 3′-terminal nucleotide of the nucleic acid molecule and/or toa nucleotide of the nucleic acid molecule between the 5′-terminalnucleotide of the nucleic acid molecule and the 3′-terminal nucleotideof the nucleic acid molecule.
 15. The method of claim 1, wherein saidsubject is an animal or a human.
 16. The method of claim 1, wherein saidrespiratory disorder comprises acute lung injury and/or acuterespiratory distress syndrome.
 17. The method of claim 1, wherein saidsubject has sepsis.
 18. The method of claim 1, wherein said respiratorydisorder comprises pneumonia.
 19. The method of claim 1, wherein saidcancer comprises breast cancer, prostate cancer, ovary cancer,osteosarcoma, glioblastoma, melanoma, small cell lung cancer orcolorectal cancer.